Gene silencing

ABSTRACT

The present invention relates to unique strategies and constructs for producing a nucleic acid product that downregulates or prevents expression of a desired target polynucleotide.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. Ser. No. 11/662,872, filed Oct.15, 2007, which is a U.S. National Stage of PCT/US2005/033992, filedSep. 23, 2005, which claims priority to U.S. provisional applicationSer. Nos. 60/612,638, filed on Sep. 24, 2004, 60/619,959, filed on Oct.20, 2004, 60/653,609, filed on Feb. 16, 2005, and 60/668,071, filed onApr. 5, 2005, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to unique constructs for producing anucleic acid product that downregulates or prevents expression of adesired target polynucleotide.

BACKGROUND OF THE INVENTION

Suppression of gene expression may be accomplished by constructs thattrigger post-transcriptional or transcriptional gene silencing. Thesesilencing mechanisms may downregulate desired polynucleotide or geneexpression by chromatin modification, RNA cleavage, translationalrepression, or via hitherto unknown mechanisms. See Meister G. andTuschl T., Nature, vol. 431, pp. 343-349, 2004.

A construct that is typically used in this regard contains a desiredpolynucleotide, which shares sequence identity with at least part of atarget gene that is operably linked to a promoter and a terminator. Asis well appreciated, the promoter initiates transcription, while theterminator ends transcription at a specific site and subsequentlymediates polyadenylation. Such transcript processing is important forstability of the transcript and its transport from the nucleus and intothe cytoplasm.

In this regard, the terminator plays an important role in conventionalgene silencing constructs. For instance, WO 99/53050 describes aconstruct that comprises a promoter, a polynucleotide comprising a firstsequence with homology to a target gene and a second sequence that isinverse complementary to the target gene, and a terminator. A terminatorof conventional constructs does not necessarily have to be positionedimmediately downstream from the desired polynucleotide. For instance,Mette and co-workers described a plasmid that contains a desiredpolynucleotide that is separated from an operably linked terminator by ahygromycin gene (Mette et al., EMBO J 18: 241-8, 1999; Mette et al.,EMBO J 19: 5194-201, 2000).

Other conventional constructs designed to silence genes contain apolynucleotide in the sense or antisense orientation between promoterand terminator. Such a conventional gene silencing construct typicallyproduces RNA transcripts that are similar in size, determined by thedistance from transcription start to termination cleavage site and thepoly-adenylated tail.

The present invention relates to new strategies and constructs for genesilencing that are generally more effective than conventionalconstructs. Furthermore, the present invention relates to new strategiesand constructs for gene silencing using a polynucleotide that is notoperably linked to a promoter and a terminator but is instead operablylinked to two convergently-oriented promoters.

SUMMARY OF THE INVENTION

Strategies and constructs of the present invention can be characterizedby certain features. A construct of the present invention, for instance,may not comprise a DNA region, such as a terminator, that is involved in3′-end formation and polyadenylation. Alternatively, the construct maycomprise a non-functional terminator that is naturally non-functional orwhich has been modified or mutated to become non-functional.

A construct may also be characterized in the arrangement of promoters ateither side of a desired polynucleotide. Hence, a construct of thepresent invention may comprise two or more promoters which flank one ormore desired polynucleotides or which flank copies of a desiredpolynucleotide, such that both strands of the desired polynucleotide aretranscribed. That is, one promoter may be oriented to initiatetranscription of the 5′-end of a desired polynucleotide, while a secondpromoter may be operably oriented to initiate transcription from the3′-end of the same desired polynucleotide. The oppositely-orientedpromoters may flank multiple copies of the desired polynucleotide.Hence, the “copy number” may vary so that a construct may comprise 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100, ormore than 100 copies, or any integer in-between, of a desiredpolynucleotide ultimately flanked by promoters that are oriented toinduce convergent transcription.

Alternatively, a first promoter may be operably linked to a firstpolynucleotide in “cassette A,” for instance, and a second promoter maybe operably linked to a second polynucleotide, e.g., “cassette B.” Thepolynucleotides of each cassette may or may not comprise the samenucleotide sequence, but may share some percentage of sequence identitywith a target nucleic acid of interest. The cassettes may be tandemlyarranged, i.e., so that they are adjacent to one another in theconstruct. Furthermore, cassette B, for instance, may be oriented in theinverse complementary orientation to cassette A. In this arrangement,therefore, transcription from the promoter of cassette B will proceed inthe direction toward the promoter of cassette A. Hence, the cassettesare arranged to induce “convergent transcription.”

If neither cassette comprises a terminator sequence, then such aconstruct, by virtue of the convergent transcription arrangement, mayproduce RNA transcripts that are of different lengths.

In this situation, therefore, there may exist subpopulations ofpartially or fully transcribed RNA transcripts that comprise partial orfull-length sequences of the transcribed desired polynucleotide from therespective cassette. Alternatively, in the absence of a functionalterminator, the transcription machinery may proceed past the end of adesired polynucleotide to produce a transcript that is longer than thelength of the desired polynucleotide.

In a construct that comprises two copies of a desired polynucleotide,therefore, where one of the polynucleotides may or may not be orientedin the inverse complementary direction to the other, and where thepolynucleotides are operably linked to promoters to induce convergenttranscription, and there is no functional terminator in the construct,the transcription machinery that initiates from one desiredpolynucleotide may proceed to transcribe the other copy of the desiredpolynucleotide and vice versa. The multiple copies of the desiredpolynucleotide may be oriented in various permutations: in the casewhere two copies of the desired polynucleotide are present in theconstruct, the copies may, for example, both be oriented in samedirection, in the reverse orientation to each other, or in the inversecomplement orientation to each other, for example.

In an arrangement where one of the desired polynucleotides is orientedin the inverse complementary orientation to the other polynucleotide, anRNA transcript may be produced that comprises not only the “sense”sequence of the first polynucleotide but also the “antisense” sequencefrom the second polynucleotide. If the first and second polynucleotidescomprise the same or substantially the same DNA sequences, then thesingle RNA transcript may comprise two regions that are complementary toone another and which may, therefore, anneal. Hence, the single RNAtranscript that is so transcribed, may form a partial or full hairpinduplex structure.

On the other hand, if two copies of such a long transcript wereproduced, one from each promoter, then there will exist two RNAmolecules, each of which would share regions of sequence complementaritywith the other. Hence, the “sense” region of the first RNA transcriptmay anneal to the “antisense” region of the second RNA transcript andvice versa. In this arrangement, therefore, another RNA duplex may beformed which will consist of two separate RNA transcripts, as opposed toa hairpin duplex that forms from a single self-complementary RNAtranscript.

Alternatively, two copies of the desired polynucleotide may be orientedin the same direction so that, in the case of transcriptionread-through, the long RNA transcript that is produced from one promotermay comprise, for instance, the sense sequence of the first copy of thedesired polynucleotide and also the sense sequence of the second copy ofthe desired polynucleotide. The RNA transcript that is produced from theother convergently-oriented promoter, therefore, may comprise theantisense sequence of the second copy of the desired polynucleotide andalso the antisense sequence of the first polynucleotide. Accordingly, itis likely that neither RNA transcript would contain regions of exactcomplementarity and, therefore, neither RNA transcript is likely to foldon itself to produce a hairpin structure. On the other hand the twoindividual RNA transcripts could hybridize and anneal to one another toform an RNA duplex.

Hence, in one aspect, the present invention provides a construct thatlacks a terminator or lacks a terminator that is preceded byself-splicing ribozyme encoding DNA region, but which comprises a firstpromoter that is operably linked to a first polynucleotide and a secondpromoter that is operably linked to second polynucleotide, whereby (1)the first and second polynucleotide share at least some sequenceidentity with each other, (2) the first promoter is oriented such thatthe direction of transcription initiated by this promoter proceedstowards the second promoter, and vice versa, and (3) this convergentarrangement produces a range of RNA transcripts that are generallydifferent in length.

The desired polynucleotides may be perfect or imperfect repeats of oneanother, or perfect or imperfect inverse complementary repeats of oneanother. In the case of a construct that comprises a firstpolynucleotide and a second polynucleotide, the second polynucleotidemay be fully or partially identical in nucleotide sequence to the firstpolynucleotide and oriented in the direct or inverse complementaryorientation with respect to the first polynucleotide. Hence, the firstand second polynucleotides may be perfect repeats of one another. On theother hand, the second polynucleotide may be an imperfect repeat of thefirst polynucleotide, that is the second polynucleotide may sharesequence identity with the first polynucleotide, but is not fully orpartially identical in sequence, i.e., the second polynucleotide is animperfect repeat. That second polynucleotide also may be oriented as adirect repeat or positioned in the inverse complementary orientationwith respect to the first polynucleotide.

Any of the polynucleotides described herein, such as a desiredpolynucleotide, or a first or second polynucleotide, for instance, maybe identical to at least a part of a target sequence, or may sharesequence identity with at least a part of a target sequence. When adesired polynucleotide comprises a sequence that is homologous to afragment of a target sequence, i.e., it shares sequence identity with“at least a part of” a target sequence, then it may be desirable thatthe nucleotide sequence of the fragment is specific to the target gene,and/or the partial perfect or imperfect sequence of the target that ispresent in the desired polynucleotide is of sufficient length to confertarget-specificity. Hence the portion of the desired polynucleotide thatshares sequence identity with a part of a target sequence may comprise acharacteristic domain, binding site, or nucleotide sequence typicallyconserved by isoforms or homologs of the target sequence. It ispossible, therefore, to design a desired polynucleotide that is optimalfor targeting a target nucleic acid in a cell.

In another embodiment, the desired polynucleotide comprises a sequenceof preferably between 4 and 5,000 nucleotides, more preferably between50 and 1,000 nucleotides, and most preferably between 150 and 500nucleotides that share sequence identity with the DNA or RNA sequence ofa target nucleic acid. The desired polynucleotide may share sequenceidentity with at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 300, 400, 500, or more than 500 contiguousnucleotides, or any integer in between, that are 100% identical insequence with a sequence in a target sequence, or a desiredpolynucleotide comprises a sequence that shares about 99%, 98%, 97%,96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%,68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%,54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%,40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 8%, 27%,26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% nucleotide sequenceidentity with a sequence of the target sequence. In other words thedesired polynucleotide may be homologous to or share homology with thefull-length sequence of a target sequence or a fragment thereof of atarget sequence.

Hence, the present invention provides an isolated nucleic acid moleculecomprising a polynucleotide that shares homology with a target sequenceand which, therefore, may hybridize under stringent or moderatehybridization conditions to a portion of a target sequence describedherein. By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides, and more preferably atleast about 20 nucleotides, and still more preferably at least about 30nucleotides, and even more preferably more than 30 nucleotides of thereference polynucleotide. For the purpose of the invention, twosequences that share homology, i.e., a desired polynucleotide and atarget sequence, may hybridize when they form a double-stranded complexin a hybridization solution of 6×SSC, 0.5% SDS, 5×Denhardt's solutionand 100 μg of non-specific carrier DNA. See Ausubel et al., section 2.9,supplement 27 (1994). Such sequence may hybridize at “moderatestringency,” which is defined as a temperature of 60° C. in ahybridization solution of 6×SSC, 0.5% SDS, 5×Denhardt's solution and 100μg of non-specific carrier DNA. For “high stringency” hybridization, thetemperature is increased to 68° C. Following the moderate stringencyhybridization reaction, the nucleotides are washed in a solution of2×SSC plus 0.05% SDS for five times at room temperature, with subsequentwashes with 0.1×SSC plus 0.1% SDS at 60° C. for lh. For high stringency,the wash temperature is increased to typically a temperature that isabout 68° C. Hybridized nucleotides may be those that are detected using1 ng of a radiolabeled probe having a specific radioactivity of 10,000cpm/ng, where the hybridized nucleotides are clearly visible followingexposure to X-ray film at −70° C. for no more than 72 hours.

In one embodiment, a construct of the present invention may comprise anexpression cassette that produces a nucleic acid that reduces theexpression level of a target gene that is normally expressed by a cellcontaining the construct, by 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%,77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%,63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%,49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%,35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%,21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1% in comparison to a cell that does not contain theconstruct.

Any polynucleotide of the present invention, be it a “desiredpolynucleotide,” a “first” polynucleotide, a “second” polynucleotide mayshare a certain percentage sequence identity with a target sequence. Asexplained herein, a target sequence may be, but is not limited to, asequence, partial or full-length, of a gene, regulatory element, such asa promoter or terminator, exon, intron, an untranslated region, or anysequence upstream or downstream of a target genomic sequence.Accordingly, a polynucleotide of the present invention, may comprise asequence that is identical over the length of that sequence to such atarget sequence. On the other hand, the polynucleotide of the presentinvention, may comprise a sequence that shares sequence identity to sucha target sequence. Hence, a desired polynucleotide of the presentinvention may share about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%,76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,62%, 61%, or 60% nucleotide sequence identity with a sequence of thetarget sequence.

In another embodiment, a desired polynucleotide comprises a sequencethat is derived from a target promoter. The target promoter may eitherbe naturally present in a cell genome, that is, the target promoter isendogenous to the cell genome, or may be introduced into that genomethrough transformation. The derived promoter of the polynucleotide maybe functionally active and contain a TATA box or TATA box-like sequencebut neither the transcription start nor any transcribed sequences beyondthe transcription start. Alternatively, the derived promoter of thepolynucleotide may be functionally inactive by, for instance, theabsence of a TATA box. Such a derived promoter may represent only partof the target promoter.

In another embodiment, the desired polynucleotide comprises a sequencethat is specific to an intron that is endogenous to a cell genome.

In another embodiment, the desired polynucleotide comprises a sequencethat is part of a terminator that is endogenous to a cell genome.

In another embodiment, the construct comprises two identical promotersthat are functionally active in a target tissue. In another embodiment,the construct comprises two different promoters, each of which isfunctionally active in a target tissue.

A construct of the present invention may further comprise one or moreadditional polynucleotides between cassette A and cassette B. Forinstance, in the 5′- to 3′-orientation, a construct may comprise (i) afirst promoter, (ii) a desired polynucleotide, (iii) an additionalpolynucleotide spacer, e.g., an intron, (iv) the inverse complement copyof the desired polynucleotide, and (v) a second promoter, where thefirst and second promoters are operably linked to the desiredpolynucleotide and the complementary copy, respectively, and areoriented to induce convergent transcription.

The additional spacer polynucleotide may be of any length. That is, thespacer polynucleotide may be an intron that is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 300, 400, 500, or more than 500 nucleotides, or anyinteger in between in length. If the spacer polynucleotide between twodesired polynucleotides is long enough, transcription may never proceedfrom promoter to the other. That is, for whatever reason, transcriptionmay stop whilst the transcription machinery is located in the spacerthat does not contain a functionally active terminator element.Accordingly, the resultant transcript may comprise the full-lengthsequence of a first desired polynucleotide and a partial sequence of theintron, but no part of the second desired polynucleotide. Thus, it maybe possible to design a construct as described herein with a spacerpolynucleotide that prevents transcription from proceeding from onedesired polynucleotide to the other. In such a situation, and if one ofthe desired polynucleotides is oriented as the inverse complementarycopy of the other, then the prevention of transcription read-throughwould, therefore, avoid the synthesis of an RNA transcript that isself-complementary.

Accordingly, depending on any of (i) the convergent arrangement ofpromoters and desired polynucleotides, (ii) the copy number of thedesired polynucleotides, (iii) the absence of a terminator region fromthe construct, and (iv) the complementarity and length of the resultanttranscripts, various populations of RNA molecules may be produced fromthe present constructs.

Hence, a single construct of the present invention may produce (i) asingle stranded “sense” RNA transcript, (ii) a single-stranded“antisense” RNA transcript, (iii) a hairpin duplex formed by asingle-stranded RNA transcript that anneals to itself, or (iv) an RNAduplex formed from two distinct RNA transcripts that anneal to eachother. A single construct may be designed to produce only sense or onlyantisense RNA transcripts from each convergently-arranged promoter.

The present invention also provides a method of reducing expression of agene normally capable of being expressed in a plant cell, by stablyincorporating any of the constructs described herein into the genome ofa cell.

In this regard, any type of cell from any species may be exposed to orstably- or transiently-transformed with a construct of the presentinvention. Hence, a bacterial cell, viral cell, fungal cell, algae cell,worm cell, plant cell, insect cell, reptile cell, bird cell, fish cell,or mammalian cell may be transformed with a construct of the presentinvention. The target sequence, therefore, may be located in the nucleusor a genome of any on of such cell types. The target sequence,therefore, may be located in a gene in the cell genome. Hence, thetarget sequence may be located in at least one of a regulatory elementof the gene, an exon of the gene, an intron of the gene, the5′-untranslated region of the gene, or the 3′-untranslated region of thegene. In one embodiment, the regulatory element of the gene is at leastone of the promoter or an enhancer element of the gene.

Alternatively, the target sequence may be located in an RNA transcriptthat is present in one of these cells and which may or may not benormally produced by the cell. That is, the RNA transcript thatcomprises the target sequence may be produced from a source that isforeign to the host cell. For instance, the RNA transcript thatcomprises the target sequence may be of viral origin but exists in aplant cell.

The present invention also contemplates in vitro, ex vivo, ex planta andin vivo exposure and integration of the desired construct into a cellgenome or isolated nucleic acid preparations.

The constructs of the present invention, for example, may be insertedinto Agrobacterium-derived transformation plasmids that containrequisite T-DNA border elements for transforming plant cells.Accordingly, a culture of plant cells may be transformed with such atransformation construct and, successfully transformed cells, grown intoa desired transgenic plant that expresses the convergently operatingpromoter/polynucleotide cassettes.

The promoters may be constitutive or inducible promoters or permutationsthereof “Strong” promoters, for instance, can be those isolated fromviruses, such as rice tungro bacilliform virus, maize streak virus,cassava vein virus, mirabilis virus, peanut chlorotic streakcaulimovirus, figwort mosaic virus and chlorella virus. Other promoterscan be cloned from bacterial species such as the promoters of thenopaline synthase and octopine synthase gene. There are variousinducible promoters, but typically an inducible promoter can be atemperature-sensitive promoter, a chemically-induced promoter, or atemporal promoter. Specifically, an inducible promoter can be a Hahsp17.7 G4 promoter, a wheat wcs 120 promoter, a Rab 16A gene promoter,an α-amylase gene promoter, a pin2 gene promoter, or a carboxylasepromoter.

Another aspect of the present invention is a construct, comprising anexpression cassette which comprises (i) a first promoter operably linkedto a first polynucleotide and (ii) a second promoter operably linked toa second polynucleotide, wherein (a) neither the first nor the secondpolynucleotide is operably linked to a terminator, (b) at least part ofthe second polynucleotide is substantially identical in nucleotidesequence to at least part of the sequence of the first polynucleotidebut is positioned within the cassette in a different orientation to thefirst polynucleotide, and (c) the direction of transcription initiatedfrom the first promoter is toward the second promoter and the directionof transcription initiated from the second promoter is toward the firstpromoter.

In one embodiment, at least part of the second polynucleotide isoriented as an inverse complement copy of at least part of the firstpolynucleotide.

In another embodiment, the sequence that terminates transcription, towhich neither polynucleotide is operably linked, is a sequence at the3′-end of a gene that is involved in 3′-end formation andpolyadenylation of the transcript of that gene.

In a preferred embodiment, the sequence that is involved in 3-endformation and polyadenylation is a terminator.

In another embodiment, the expression cassette does not comprise (i) anos gene terminator, (ii) the 3′ untranslated sequence of T-DNA gene 7,(iii) the 3′ untranslated sequences of the major inclusion body proteingene of cauliflower mosaic virus, (iv) the 3′ untranslated sequences ofthe pea ribulose 1,5-bisphosphate carboxylase small subunit, (v) the 3′untranslated sequences of the potato ubiquitin-3 gene, or (vi) the 3′untranslated sequences of the potato proteinase inhibitor II gene, (vii)the 3′ untranslated sequences of opine genes, (viii) the 3′ untranslatedsequences of endogenous genes.

In one embodiment, the first polynucleotide comprises a sequence thatshares sequence identity with a target gene or at least one of aregulatory element that is associated with the target gene, an exon ofthe target gene, an intron of the target gene, the 5′-untranslatedregion of the target gene, or the 3′-untranslated region of the targetgene.

In another embodiment, the first polynucleotide comprises a sequencethat shares about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%,88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%,74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%,60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%,46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%,32%, 31%, 30%, 29%, 8%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1% nucleotide sequence identity with a sequence of the targetsequence.

In one embodiment, the target gene is a COMT gene involved in ligninbiosynthesis, a CCOMT gene involved in lignin biosynthesis, any othergene involved in lignin biosynthesis, an R1 gene involved in starchphosphorylation, a phosphorylase gene involved in starchphosphorylation, a PPO gene involved in oxidation of polyphenols, apolygalacturonase gene involved in pectin degradation, a gene involvedin the production of allergens, a gene involved in fatty acidbiosynthesis such as FAD2.

In another embodiment, (a) the regulatory element of the target gene isthe promoter or an enhancer element associated with the target gene or(b) the first polynucleotide comprises a sequence that shares sequenceidentity with an intron of a target gene, wherein the intron comprisesthe sequence of SEQ ID NO: 44.

In a particular embodiment, the target gene is located in the genome ofa cell. Hence, the cell may be a cell from a bacteria, virus, fungus,yeast, plant, reptile, bird, fish, or mammal.

In one embodiment, the target sequence is located in a DNA sequence thatencodes an RNA transcript.

In another embodiment, the first and second promoters are functional ina plant.

In a preferred embodiment, the expression cassette is located betweentransfer-DNA border sequences of a plasmid that is suitable forbacterium-mediated plant transformation.

In yet another embodiment, the bacterium is Agrobacterium, Rhizobium, orPhyllobacterium. In one embodiment, the bacterium is Agrobacteriumtumefaciens, Rhizobium trifolii, Rhizobium leguminosarum,Phyllobacterium myrsinacearum, SinoRhizobium meliloti, and MesoRhizobiumloti.

In one embodiment, the construct further comprises a spacerpolynucleotide positioned between the first and second polynucleotides.The spacer polynucleotide may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 300, 400, 500, or more than 500 nucleotides long.

In another embodiment, the first promoter is a near-constitutivepromoter, a tissue-specific promoter, or an inducible promoter andwherein the second promoter is a near-constitutive promoter, atissue-specific promoter, or an inducible promoter.

In a particular embodiment, the constitutive strong promoter is selectedfrom the group consisting of a potato ubiquitin-7 promoter, a potatoubiquitin-3 promoter, a tomato ubiquitin promoter, an alfalfa petEpromoter, an alfalfa Pal promoter, a canola napin promoter, a maizeubiquitin promoter, a rice ubiquitin promoter, a sugarcane ubiquitinpromoter, a rice actin promoter, a rubisco small subunit promoter, and arubisco activase promoter.

In one embodiment, the tissue-specific promoter is a granule-boundstarch synthase promoter or an ADP glucose pyrophosphorylase genepromoter.

In one embodiment, the inducible promoter is a temperature-sensitivepromoter, a chemically-induced promoter, or a temporal promoter.

In one embodiment, the inducible promoter is selected from the groupconsisting of an Ha hsp17.7 G4 promoter, a wheat wcs120 promoter, a Rab16A gene promoter, an α-amylase gene promoter, a pin2 gene promoter, anda carboxylase promoter.

Another aspect of the present invention is a transformation plasmid,comprising an expression cassette, which comprises in the 5′ to 3′orientation (1) a first promoter that is operably linked to (2) a firstdesired polynucleotide, which abuts (3) at least one optional spacerpolynucleotide, where the 3′-end of one of the spacer polynucleotidesabuts a (4) a second desired polynucleotide, which is operably linked to(5) a second promoter, wherein neither desired polynucleotide in theexpression cassette is operably linked to any known transcriptionterminator.

In one embodiment, at least part of the first desired polynucleotide isin the antisense orientation and wherein at least part of the seconddesired polynucleotide is oriented as the inverse complement of thefirst desired polynucleotide.

In another embodiment, at least part of the first desired polynucleotideis in the sense orientation and wherein at least part of the seconddesired polynucleotide is oriented as the inverse complement of thefirst desired polynucleotide.

In another embodiment, at least part of the first desired polynucleotideis a promoter sequence.

In a further embodiment, the promoter sequence is from a promoterselected from the group consisting of (1) a starch-associated R1 genepromoter, (2) a polyphenol oxidase gene promoter, (3) a fatty aciddesaturase 12 gene promoter, (4) a microsomal omega-6 fatty aciddesaturase gene promoter, (5) a cotton stearoyl-acyl-carrier proteindelta 9-desaturase gene promoter, (6) an oleoyl-phosphatidylcholineomega 6-desaturase gene promoter, (7) a Medicago truncatula caffeicacid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) gene promoter,(8) a Medicago sativa (alfalfa) caffeic acid/5-hydroxyferulic acid3/5-O-methyltransferase (COMT) gene promoter, (9) a Medicago truncatulacaffeoyl CoA 3-O-methyltransferase (CCOMT) gene promoter, (10) aMedicago sativa (alfalfa) caffeoyl CoA 3-O-methyltransferase (CCOMT)gene promoter, (11) a major apple allergen Mal d 1 gene promoter, (12) amajor peanut allergen Ara h 2 gene promoter, (13) a major soybeanallergen Gly m Bd 30 K gene promoter, and (14) a polygalacturonase genepromoter.

In one embodiment, (i) at least one of the first and second promoters isa GBSS promoter, and (ii) the first desired polynucleotide is a sequencefrom a polyphenol oxidase gene.

In another embodiment, the first and second promoters are GBSSpromoters.

In one embodiment, both the first promoter is a GBSS promoter and thesecond promoter is an AGP promoter.

Another aspect of the present invention is a method of reducingexpression of a gene normally capable of being expressed in a plantcell, comprising exposing a plant cell to any construct describedherein, wherein the construct is maintained in a bacterium strain,wherein the desired polynucleotide comprises a sequence that sharessequence identity to a target sequence in the plant cell genome.

In one embodiment, the bacterium strain is Agrobacterium tumefaciens,Rhizobium trifolii, Rhizobium leguminosarum, Phyllobacteriummyrsinacearum, SinoRhizobium meliloti, and MesoRhizobium loti.

Another aspect of the present invention is a construct, comprising anexpression cassette which comprises in the 5′ to 3′ orientation (i) afirst promoter, (ii) a first polynucleotide that comprises a sequencethat shares sequence identity with at least a part of a promotersequence of a target gene, (iii) a second polynucleotide comprising asequence that shares sequence identity with the inverse complement of atleast part of the promoter of the target gene, and (iv) a secondpromoter, wherein the first promoter is operably linked to the 5′-end ofthe first polynucleotide and the second promoter is operably linked tothe 3′-end of the second polynucleotide.

Another aspect of the present invention is a construct, comprising anexpression cassette which comprises in the 5′ to 3′ orientation (i) afirst promoter, (ii) a first polynucleotide that comprises a sequencethat shares sequence identity with at least a part of a promotersequence of a target gene, (iii) a second polynucleotide comprising asequence that shares sequence identity with the inverse complement of atleast part of the promoter of the target gene, (iv) a terminator,wherein the first promoter is operably linked to the 5′-end of the firstpolynucleotide and the second polynucleotide is operably linked to theterminator.

Another aspect of the present invention is a plant transformationplasmid, comprising the sequence depicted in SEQ ID NO: 40 or 42.

Another aspect of the present invention is a method for reducingcold-induced sweetening in a tuber, comprising expressing any constructdescribed herein in a cell of a tuber, wherein (a) the firstpolynucleotide comprises the sequence of part of an R1 gene, (b) thesecond polynucleotide is the inverse complement of the firstpolynucleotide compared to the first polynucleotide, (c) one or both ofthe first and second promoters are GBSS or AGP, and (d) expression ofthe construct in the cell reduces transcription and/or translation of anR1 gene in the tuber cell genome, thereby reducing cold-inducedsweetening in the tuber. In one embodiment, the first polynucleotidecomprises the sequence depicted in SEQ ID NO: 23 or 24. In anotherembodiment, the tuber is a potato. In another embodiment, the firstpolynucleotide comprises two copies of the sequence of SEQ ID NO: 23 or24.

Another aspect of the present invention is a method for enhancingtolerance to black spot bruising in a tuber, comprising expressing anyconstruct described herein in a cell of a tuber, wherein (a) the firstpolynucleotide comprises the sequence of part of a polyphenol oxidasegene, (b) the second polynucleotide is the inverse complement of thefirst polynucleotide, (c) one or both of the first and second promotersare GBSS or AGP, and (d) expression of the construct in the cell reducestranscription and/or translation of a polyphenol oxidase gene in thetuber cell genome, thereby enhancing the tolerance of the tuber to blackspot bruising. In one embodiment, the first polynucleotide comprises thesequence of SEQ ID NO: 26 or 27. In another embodiment, the tuber is apotato. In another embodiment, the first polynucleotide comprises twocopies of the sequence of SEQ ID NO: 26 or 27.

Another aspect of the present invention is a method for increasing oleicacid levels in an oil-bearing plant, comprising expressing any constructdescribed herein in a cell of a seed of an oil-bearing plant, wherein(a) the first polynucleotide comprises the sequence of part of a Fad2gene, (b) the second polynucleotide is the inverse complement of thefirst polynucleotide, (c) one or both of the first and second promotersare napin gene, Fad2 gene, or stearoyl-ACP desaturase gene promoters,and (d) expression of the construct in the cell reduces transcriptionand/or translation of a Fad2 gene in the cell of the seed of theoil-bearing plant, thereby increasing the oil content of the seed. Inone embodiment, the first polynucleotide comprises the sequence depictedin SEQ ID NO: 28. In another embodiment, the sequence of the napin genepromoter comprises the sequence depicted in SEQ ID NO: 30.

In one embodiment, the sequence of the stearoyl-ACP desaturase genepromoter comprises the sequence depicted in SEQ ID NO: 31.

In another embodiment, the sequence of the Fad2 gene promoter comprisesthe sequence depicted in SEQ ID NO: 32.

In one embodiment, the oil-bearing plant is a Brassica plant, canolaplant, soybean plant, cotton plant, or a sunflower plant.

Another aspect of the present invention is a method for reducing lignincontent in a plant, comprising expressing any construct described hereinin a cell of the plant, wherein (a) the first polynucleotide comprisesthe sequence of part of a caffeic acid/5-hydroxyferulic acid3/5-O-methyltransferase (COMT) gene, (b) the second polynucleotide isthe inverse complement of the first polynucleotide, (c) one or both ofthe first and second promoters are petE or Pal gene promoters, and (d)expression of the construct in the cell reduces transcription and/ortranslation of a COMT gene in the cell of the plant, thereby reducinglignin content in a plant. In one embodiment, the cell is in thevascular system of the plant. In a preferred embodiment, the plant is analfalfa plant. In another embodiment, the first polynucleotide comprisesthe sequence depicted in SEQ ID NO: 33 or 37.

Another aspect of the present invention is a method for reducing thedegradation of pectin in a fruit of a plant, comprising expressing anyconstruct described herein in a fruit cell of the plant, wherein (a) thefirst polynucleotide comprises the sequence of part of polygalacturonasegene, (b) the second polynucleotide is the inverse complement of thefirst polynucleotide, (c) both of the first and second promoters arefruit-specific promoters, and (d) expression of the construct in thefruit cell reduces transcription and/or translation of apolygalacturonase gene in the cell of the plant, thereby reducing thedegradation of pectin in the fruit. In one embodiment, the firstpolynucleotide comprises the sequence depicted in SEQ ID NO: 39.

Another aspect of the present invention is a method for reducing theallergenicity of a food produced by a plant, comprising expressing anyconstruct described herein in a cell of a plant, wherein (a) the firstpolynucleotide comprises the sequence of part of a gene that encodes anallergen, (b) the second polynucleotide is the inverse complement of thefirst polynucleotide, and (c) the expression of the construct reducestranscription and/or translation of the allergen, thereby reducing theallergenicity of a food produced by the plant.

In one embodiment, (a) the plant is an apple plant, (b) the food is anapple, (c) the first polynucleotide comprises a sequence from the Mal dI gene promoter, and (d) expression of the construct in the apple plantreduces transcription and/or translation of Mal d I in the apple.

In another embodiment, (a) the plant is a peanut plant, (b) the food isa peanut, (c) the first polynucleotide comprises a sequence from the Arah 2 gene promoter, and (d) expression of the construct in the peanutplant reduces transcription and/or translation of Ara h 2 in the peanut.

In another embodiment, (a) the plant is a soybean plant, (b) the food isa soybean, (c) the first polynucleotide comprises a sequence from theGly m Bd gene promoter, and (d) expression of the construct in thesoybean plant reduces transcription and/or translation of Gly m Bd inthe soybean.

Another aspect of the present invention is a method for downregulatingthe expression of multiple genes in a plant, comprising expressing in acell of a plant a construct comprising the sequence depicted in SEQ IDNO: 40, which downregulates expression of polyphenol oxidase,phosphorylase L gene, and the R1 gene in the plant cell.

Another aspect of the present invention is a method for downregulatingthe expression of multiple genes in a plant, comprising expressing in acell of a plant a construct comprising the sequence depicted in SEQ IDNO: 42, which downregulates expression of polyphenol oxidase,phosphorylase L gene, and the R1 gene in the plant cell.

Another aspect of the present invention is a construct, comprising adesired promoter that is operably linked to (i) a first promoter at its5′-end and (ii) a second promoter at its 3′-end, wherein the desiredpromoter shares sequence identity with a target promoter in a genome ofinterest.

Another aspect of the present invention is a construct, comprising a twoconvergently-oriented copies of a desired promoter that are separated bya polynucleotide, wherein the desired promoter shares sequence identitywith a target promoter in a desired genome of interest. In oneembodiment, the polynucleotide that separates the convergently-orientedpromoters is an intron.

Another aspect of the present invention is a construct, comprising twodesired promoters that are operably linked to a promoter and aterminator, wherein the desired promoters share sequence identity with atarget promoter in a genome of interest. In one embodiment, the twodesired promoters share, over at least a part of their respectivelengths, sequence identity with each other and where one of the desiredpromoters is oriented as the inverse complement of the other.

In another aspect is a construct, comprising two desired promoters thatare operably linked to a promoter and a terminator, wherein the desiredpromoters share sequence identity with a target promoter in a genome ofinterest. In one embodiment, the two desired promoters share, over atleast a part of their respective lengths, sequence identity with eachother and where one of the desired promoters is oriented as the inversecomplement of the other.

In another aspect a construct is provided that comprises four directrepeats of a polynucleotide of interest, which are preceded by anantisense DNA fragment of the polynucleotide of interest. Such aconstruct is depicted by pSIM1111.

The present invention also provides a method for reducing the expressionlevel of an endogenous gene in an alfalfa plant, comprising introducinga cassette into an alfalfa cell, wherein the cassette comprises twoalfalfa-specific promoters arranged in a convergent orientation to eachother, wherein the activity of the promoters in the cassette reduces theexpression level of an endogenous alfalfa gene, which is operably linkedin the alfalfa genome to a promoter that has a sequence that sharessequence identity with at least a part of one of the promoters in thecassette. In one embodiment, the sequence of at least one of thepromoters is depicted in SEQ ID NO: 54 or SEQ ID NO: 55.

The present invention also provides a method for reducing the expressionof a Comt gene, comprising expressing a Comt gene fragment or Comtpromoter fragment in a cell that comprises a Comt gene in its genome.

The present invention also provides a method for reducing the expressionof a Comt gene or Ccomt gene, comprising expressing the construct of anyconstruct described herein in a cell that comprises a Comt gene or aCcomt gene in its genome, wherein the first polynucleotide comprises asequence of a Comt gene or Comt gene promoter or a Ccomt gene or Ccomtgene promoter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts schematic diagrams for T-DNAs of binary vectors that (a)represent a negative control (pSIM714), and (b) comprise constructs thatrepresent conventional silencing constructs, pSIM374, pSIM718, andpSIM755. “B” denotes a transfer-DNA border sequence; “T” denotes aterminator sequence; “hptII” is a resistance gene that confershygromycin resistance to a plant; “P1” denotes a promoter sequence and,in this example, is a promoter that is identical to the promoter drivinga functionally active bete-glucuronidase (gus) gene in the transgenicgus plant; “P2” denotes a promoter sequence that is also functionallyactive but different from P1; “gus-S” denotes a gus gene fragment;“gus-A” denotes an inverse complement of the gus gene fragment; “I”denotes an intron. With respect to gus-S and gus-A, the solid thickarrows signify (part of the) RNA transcripts that share identity with apart of the transcript produced by expressing the gus gene; the dottedthick arrows signify (part of the) RNA transcripts that share identitywith a part of the inverse complement of the gus gene transcript; thethin lines signify parts of the transcript with homology or inversecomplementarity to another sequence such as the intron of the construct.In this respect, the leftward pointing open arrow (which denotes the“gus-A” element in the cassette) indicates that the gus-A element isoriented in the expression cassette as the inverse complement of thegus-S, the rightward pointing arrow. Hence, P1 and P2 promoters areoriented so that transcription from each proceeds in a convergentmanner, i.e., transcription of P1 proceeds toward P2 and vice versa.

FIG. 2 depicts schematic diagrams for T-DNAs of binary vectorscomprising constructs that resemble conventional silencing constructsexcept that they lack a terminator, pSIM728, pSIM140, and pSIM758. Withrespect to gus-S and gus-A, the solid thick arrows signify the part ofthe RNA transcripts that share identity with a part of the transcriptproduced by expressing the gus gene; the dotted thick arrows signifyparts of the RNA transcripts that share identity with a part of theinverse complement of the gus gene transcript; the thin lines signifyparts of the transcript with homology or inverse complementarity toanother sequence such as the intron of the construct.

FIG. 3 depicts schematic diagrams for T-DNAs comprising “terminator-freecolliding transcription” (TFCT) constructs. Specifically, it illustratesthe T-DNAs of pSIM715, pSIM717, pSIM756, and pSIM771. The key to theidentified elements and solid and dotted arrows is the same as thoseexplained in the legend of FIG. 1. In pSIM717, read-through oftranscription originated from both P1 and P2 over the intron producestranscripts that contain 5′-sequences identical to part of the gus genetranscript and 3′-sequences that are inverse complementary to the gusgene transcript. These transcripts may fold to produce partiallydouble-stranded RNA. Depending on the ability of the P1 transcriptioncomplex to proceed unencumbered, an RNA transcript, initiated from theP1 promoter, could conceivably transcribe sequences downstream of thegus-S sequence to which it is operably linked. Accordingly, when readingthe “top,” i.e., sense strand of pSIM717, in a 5′- to 3′-direction, atranscript from P1 may comprise the sequence of the intervening intron(“I”), as well as the sequence of the inverse complement gus-S element.The “top” strand sequence of the inverse complement gus-S element is theantisense of gus-S.

FIG. 4 depicts schematic diagrams for T-DNAs comprising “terminator-freecolliding transcription” (TFCT) constructs. Specifically, it illustratesthe T-DNAs of pSIM754, pSIM773, and pSIM767. The key to the identifiedelements and solid and dotted arrows is the same as those explained inthe legend of FIG. 1. P1n indicates the part of the P1 promoter that isupstream from the TATA box. This sequence is not functional as promoter.

FIG. 5 depicts schematic diagrams for T-DNAs comprising “terminator-freecolliding transcription” (TFCT) constructs. Specifically, it illustratesthe T-DNAs of pSIM782. The key to the identified elements and solid anddotted arrows is the same as those explained in the legend of FIG. 1.gusI indicates the intronm of the gus gene.

FIG. 6 depicts schematic diagrams for T-DNAs comprising “terminator-freecolliding transcription” (TFCT) constructs. Specifically, it illustratesthe T-DNAs of pSIM765, pSIM922F, pSIM922G, pSIM774, and pSIM775. The keyto the identified elements and solid and dotted arrows is the same asthose explained in the legend of FIG. 1. PPO indicates a fragment of thetobacco PPO gene.

FIG. 7 shows ethidium bromide-stained agarose gels containing theproducts of RT-PCRs. +=positive plasmid control; −=negative control;M=marker; T1=transcript from P1 promoter; T2=transcript from P2promoter.

FIG. 8 shows autoradiograms of RNA gel blots. The probe used forhybridization was derived from the gus gene.

FIG. 9 shows a sequence analysis of the various promoter fragments andidentifies a 89-bp sequence that may be methylated during promoter-basedsilencing.

FIG. 10 depicts plasmid maps. G: gus gene fragment; H: expressioncassette for hptII gene; LB: left border region; RB: right borderregion; T: terminator; P1: P1 promoter; Pln: non-functional P1 promoterlacking a TATA box; P2: P2 promoter; P3: P3 promoter; GB: GBSS promoter;PP: PPO gene fragment; PT=fragment of tobacco PPO gene. Direction oftranscription is indicated with a small black solid arrow.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A construct of the present invention can be used to efficiently reduceor prevent the transcription or translation of a target nucleic acid bytriggering convergent transcription of a desired polynucleotide. Henceone goal of the present invention is to provide constructs that producenucleic acid molecules that prevent or reduce expression of a gene or ofa gene product, such as an RNA transcript or protein.

One particular characteristic of such a construct is that, in contrastto conventional silencing constructs, no functional terminator isinserted and operably linked to the 3′-end of a desired polynucleotide.It is well established that a terminator is a nucleotide sequence,typically located at the 3′-end of a gene, that is involved in cleavageof the RNA transcript that is transcribed from the gene and inpolyadenylation of that transcript. Typically, a terminator is locateddownstream of the gene's stop codon.

Terminators that were used for the construction of conventionalsilencing cassettes, and which are excluded from constructs of thepresent invention, were derived from such 3′-regions of certain genesand often also included even more downstream non-transcribed DNAsequences. The choice of which terminator to use has more often thannot, simply been a matter of convenience. Hence, opine terminators ortermination regions from endogenous and previously characterized geneshave been used in conventional silencing constructs. One of the morefrequently used terminators, for instance, is the Agrobacterium nopalinesynthase (nos) gene terminator, which comprises both 3′ untranslatedsequences and some additional downstream DNA. Other terminators include:

The 3′ untranslated sequences of T-DNA gene 7 (Genbank accessionV00090).

The 3′ untranslated sequences of the major inclusion body protein geneof cauliflower mosaic virus.

The 3′ untranslated sequences of the pea ribulose 1,5-bisphosphatecarboxylase small subunit (Genbank accession M21375).

The 3′ untranslated sequences of the potato ubiquitin-3 gene (Genbankaccession Z11669).

The 3′ untranslated sequences of the potato proteinase inhibitor II gene(Genbank accession CQ889094).

The 3′ untranslated sequences of opine genes.

The 3′ untranslated sequences of endogenous genes; that is genes thatare normally expressed by the genome of an organism.

With respect to the present invention, however, none of suchterminators, indeed, no functional terminator, is directly operablylinked to a desired polynucleotide of the present construct. Nor is adesired polynucleotide directly operably linked to a terminator that ispreceded by a self-splicing ribozyme-encoding sequence.

Another characteristic of the construct of the present invention is thatit promotes convergent transcription of one or more copies ofpolynucleotide that is or are not directly operably linked to aterminator, via two opposing promoters. Due to the absence of atermination signal, the length of the pool of RNA molecules that istranscribed from the first and second promoters may be of variouslengths.

Occasionally, for instance, the transcriptional machinery may continueto transcribe past the last nucleotide that signifies the “end” of thedesired polynucleotide sequence. Accordingly, in this particulararrangement, transcription termination may occur either through the weakand unintended action of downstream sequences that, for instance,promote hairpin formation or through the action of unintendedtranscriptional terminators located in plant DNA flanking the transferDNA integration site.

A terminator-free colliding transcription (TFCT) construct of thepresent invention, therefore, may comprise a first promoter operablylinked to a first polynucleotide and a second promoter operably linkedto a second polynucleotide, whereby (1) the first and secondpolynucleotides share at least some sequence identity with each otherand a target sequence, and (2) the first promoter is oriented such thatthe direction of transcription initiated by this promoter proceedstowards the second promoter, and vice versa, (3) the construct producesRNA molecules that are generally different in size, some transcriptsrepresenting the RNA counterparts of at least part of the polynucleotideand others comprising the counterparts of at least some of both thepolynucleotide and its inverse complement.

The desired polynucleotide may be linked in two different orientationsto the promoter. In one orientation, e.g., “sense”, at least the 5′-partof the resultant RNA transcript will share sequence identity with atleast part of at least one target transcript. An example of thisarrangement is shown in FIG. 3 as pSIM717. In the other orientationdesignated as “antisense”, at least the 5′-part of the predictedtranscript will be identical or homologous to at least part of theinverse complement of at least one target transcript. An example of thelatter arrangement is shown in FIG. 3 as pSIM756.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified region. When percentage of sequenceidentity is used in reference to proteins it is recognized that residuepositions which are not identical often differ by conservative aminoacid substitutions, where amino acid residues are substituted for otheramino acid residues with similar chemical properties (e.g. charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. Where sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch,J. Mol. Biol. 48: 443 (1970); by the search for similarity method ofPearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24:307-331 (1994).

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); Altschul et al., J.Mol. Biol., 215:403-410 (1990); and, Altschul et al., Nucleic Acids Res.25:3389-3402 (1997).

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5877 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequenceswhich may be homopolymeric tracts, short-period repeats, or regionsenriched in one or more amino acids. Such low-complexity regions may bealigned between unrelated proteins even though other regions of theprotein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993))and XNU (Clayerie and States, Comput. Chem., 17:191-201 (1993))low-complexity filters can be employed alone or in combination.

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

Any or all of the elements and DNA sequences that are described hereinmay be endogenous to one or more plant genomes. Accordingly, in oneparticular embodiment of the present invention, all of the elements andDNA sequences, which are selected for the ultimate transfer cassette areendogenous to, or native to, the genome of the plant that is to betransformed. For instance, all of the sequences may come from a potatogenome. Alternatively, one or more of the elements or DNA sequences maybe endogenous to a plant genome that is not the same as the species ofthe plant to be transformed, but which function in any event in the hostplant cell. Such plants include potato, tomato, and alfalfa plants. Thepresent invention also encompasses use of one or more genetic elementsfrom a plant that is interfertile with the plant that is to betransformed.

Public concerns were addressed through development of an all-nativeapproach to making genetically engineered plants, as disclosed byRommens et al. in WO2003/069980, US-2003-0221213, US-2004-0107455, andWO2005/004585, which are all incorporated herein by reference. Rommenset al. teach the identification and isolation of genetic elements fromplants that can be used for bacterium-mediated plant transformation.Thus, Rommens teaches that a plant-derived transfer-DNA (“P-DNA”), forinstance, can be isolated from a plant genome and used in place of anAgrobacterium T-DNA to genetically engineer plants.

In this regard, a “plant” of the present invention includes, but is notlimited to angiosperms and gymnosperms such as potato, tomato, tobacco,avocado, alfalfa, lettuce, carrot, strawberry, sugarbeet, cassava, sweetpotato, soybean, pea, bean, cucumber, grape, brassica, maize, turfgrass, wheat, rice, barley, sorghum, oat, oak, eucalyptus, walnut, andpalm. Thus, a plant may be a monocot or a dicot. “Plant” and “plantmaterial,” also encompasses plant cells, seed, plant progeny, propagulewhether generated sexually or asexually, and descendents of any ofthese, such as cuttings or seed. “Plant material” may refer to plantcells, cell suspension cultures, callus, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,seeds, germinating seedlings, and microspores. Plants may be at variousstages of maturity and may be grown in liquid or solid culture, or insoil or suitable media in pots, greenhouses or fields. Expression of anintroduced leader, trailer or gene sequences in plants may be transientor permanent.

Thus, any one of such plants and plant materials may be transformedaccording to the present invention. In this regard, transformation of aplant is a process by which DNA is stably integrated into the genome ofa plant cell. “Stably” refers to the permanent, or non-transientretention and/or expression of a polynucleotide in and by a cell genome.Thus, a stably integrated polynucleotide is one that is a fixture withina transformed cell genome and can be replicated and propagated throughsuccessive progeny of the cell or resultant transformed plant.Transformation may occur under natural or artificial conditions usingvarious methods well known in the art. See, for instance, METHODS INPLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Bernard R. Glick and John E.Thompson (eds), CRC Press, Inc., London (1993); Chilton, ScientificAmerican, 248)(6), pp. 36-45, 1983; Bevan, Nucl. Acids. Res., 12, pp.8711-8721, 1984; and Van Montague et al., Proc R Soc Lond B Biol Sci.,210(1180), pp. 351-65, 1980. Plants also may be transformed using“Refined Transformation” and “Precise Breeding” techniques. See, forinstance, Rommens et al. in WO2003/069980, US-2003-0221213,US-2004-0107455, WO2005/004585, US-2004-0003434, US-2005-0034188,WO2005/002994, and WO2003/079765, which are all incorporated herein byreference.

One or more traits of a tuber-bearing plant of the present invention maybe modified using the transformation sequences and elements describedherein. A “tuber” is a thickened, usually underground, food-storingorgan that lacks both a basal plate and tunic-like covering, which cormsand bulbs have. Roots and shoots grow from growth buds, called “eyes,”on the surface of the tuber. Some tubers, such as caladiums, diminish insize as the plants grow, and form new tubers at the eyes. Others, suchas tuberous begonias, increase in size as they store nutrients duringthe growing season and develop new growth buds at the same time. Tubersmay be shriveled and hard or slightly fleshy. They may be round, flat,odd-shaped, or rough. Examples of tubers include, but are not limited toahipa, apio, arracacha, arrowhead, arrowroot, baddo, bitter casava,Brazilian arrowroot, cassava, Chinese artichoke, Chinese water chestnut,coco, cocoyam, dasheen, eddo, elephant's ear, girasole, goo, Japaneseartichoke, Japanese potato, Jerusalem artichoke, jicama, lilly root,ling gaw, mandioca, manioc, Mexican potato, Mexican yam bean, oldcocoyam, potato, saa got, sato-imo, seegoo, sunchoke, sunroot, sweetcasava, sweet potatoes, tanier, tannia, tannier, tapioca root,topinambour, water lily root, yam bean, yam, and yautia. Examples ofpotatoes include, but are not limited to Russet Potatoes, Round WhitePotatoes, Long White Potatoes, Round Red Potatoes, Yellow FleshPotatoes, and Blue and Purple Potatoes.

Tubers may be classified as “microtubers,” “minitubers,” “near-mature”tubers, and “mature” tubers. Microtubers are tubers that are grown ontissue culture medium and are small in size. By “small” is meant about0.1 cm-1 cm. A “minituber” is a tuber that is larger than a microtuberand is grown in soil. A “near-mature” tuber is derived from a plant thatstarts to senesce, and is about 9 weeks old if grown in a greenhouse. A“mature” tuber is one that is derived from a plant that has undergonesenescence. A mature tuber is, for example, a tuber that is about 12 ormore weeks old.

In this respect, a plant-derived transfer-DNA (“P-DNA”) border sequenceof the present invention is not identical in nucleotide sequence to anyknown bacterium-derived T-DNA border sequence, but it functions foressentially the same purpose. That is, the P-DNA can be used to transferand integrate one polynucleotide into another. A P-DNA can be insertedinto a tumor-inducing plasmid, such as a Ti-plasmid from Agrobacteriumin place of a conventional T-DNA, and maintained in a bacterium strain,just like conventional transformation plasmids. The P-DNA can bemanipulated so as to contain a desired polynucleotide, which is destinedfor integration into a plant genome via bacteria-mediated planttransformation. See Rommens et al. in WO2003/069980, US-2003-0221213,US-2004-0107455, and WO2005/004585, which are all incorporated herein byreference.

Thus, a P-DNA border sequence is different by 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides from aknown T-DNA border sequence from an Agrobacterium species, such asAgrobacterium tumefaciens or Agrobacterium rhizogenes.

A P-DNA border sequence is not greater than 99%, 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%,80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%,66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%,52%, 51% or 50% similar in nucleotide sequence to an Agrobacterium T-DNAborder sequence.

Methods were developed to identify and isolate transfer DNAs fromplants, particularly potato and wheat, and made use of the border motifconsensus described in US-2004-0107455, which is incorporated herein byreference.

In this respect, a plant-derived DNA of the present invention, such asany of the sequences, cleavage sites, regions, or elements disclosedherein is functional if it promotes the transfer and integration of apolynucleotide to which it is linked into another nucleic acid molecule,such as into a plant chromosome, at a transformation frequency of about99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%,about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%,about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%,about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, about60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%,about 53%, about 52%, about 51%, about 50%, about 49%, about 48%, about47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%,about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%,about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about21%, about 20%, about 15%, or about 5% or at least about 1%.

Any of such transformation-related sequences and elements can bemodified or mutated to change transformation efficiency. Otherpolynucleotide sequences may be added to a transformation sequence ofthe present invention. For instance, it may be modified to possess 5′-and 3′-multiple cloning sites, or additional restriction sites. Thesequence of a cleavage site as disclosed herein, for example, may bemodified to increase the likelihood that backbone DNA from theaccompanying vector is not integrated into a plant genome.

Any desired polynucleotide may be inserted between any cleavage orborder sequences described herein. For example, a desired polynucleotidemay be a wild-type or modified gene that is native to a plant species,or it may be a gene from a non-plant genome. For instance, whentransforming a potato plant, an expression cassette can be made thatcomprises a potato-specific promoter that is operably linked to adesired potato gene or fragment thereof and a potato-specificterminator. The expression cassette may contain additional potatogenetic elements such as a signal peptide sequence fused in frame to the5′-end of the gene, and a potato transcriptional enhancer. The presentinvention is not limited to such an arrangement and a transformationcassette may be constructed such that the desired polynucleotide, whileoperably linked to a promoter, is not operably linked to a terminatorsequence.

In addition to plant-derived elements, such elements can also beidentified in, for instance, fungi and mammals. See, for instance, SEQID NOs: 173-182. Several of these species have already been shown to beaccessible to Agrobacterium-mediated transformation. See Kunik et al.,Proc Natl Acad Sci USA 98: 1871-1876, 2001, and Casas-Flores et al.,Methods Mol Biol 267: 315-325, 2004, which are incorporated herein byreference.

When a transformation-related sequence or element, such as thosedescribed herein, are identified and isolated from a plant, and if thatsequence or element is subsequently used to transform a plant of thesame species, that sequence or element can be described as “native” tothe plant genome.

Thus, a “native” genetic element refers to a nucleic acid that naturallyexists in, originates from, or belongs to the genome of a plant that isto be transformed. In the same vein, the term “endogenous” also can beused to identify a particular nucleic acid, e.g., DNA or RNA, or aprotein as “native” to a plant. Endogenous means an element thatoriginates within the organism. Thus, any nucleic acid, gene,polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is isolated eitherfrom the genome of a plant or plant species that is to be transformed oris isolated from a plant or species that is sexually compatible orinterfertile with the plant species that is to be transformed, is“native” to, i.e., indigenous to, the plant species. In other words, anative genetic element represents all genetic material that isaccessible to plant breeders for the improvement of plants throughclassical plant breeding. Any variants of a native nucleic acid also areconsidered “native” in accordance with the present invention. In thisrespect, a “native” nucleic acid may also be isolated from a plant orsexually compatible species thereof and modified or mutated so that theresultant variant is greater than or equal to 99%, 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%,80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%,66%, 65%, 64%, 63%, 62%, 61%, or 60% similar in nucleotide sequence tothe unmodified, native nucleic acid isolated from a plant. A nativenucleic acid variant may also be less than about 60%, less than about55%, or less than about 50% similar in nucleotide sequence.

A “native” nucleic acid isolated from a plant may also encode a variantof the naturally occurring protein product transcribed and translatedfrom that nucleic acid. Thus, a native nucleic acid may encode a proteinthat is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%,77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%,63%, 62%, 61%, 60% similar in amino acid sequence to the unmodified,native protein expressed in the plant from which the nucleic acid wasisolated.

In a terminator-free construct that so comprises two copies of thedesired polynucleotide, one desired polynucleotide may be oriented sothat its sequence is the inverse complement of the other. The schematicdiagram of pSIM717 in FIG. 3 illustrates such an arrangement. That is,the “top,” “upper,” or “sense” strand of the construct would comprise,in the 5′- to 3′-direction, (1) a target gene fragment, and (2) theinverse complement of a target gene fragment. In this arrangement, asecond promoter that is operably linked to that inverse complement ofthe desired polynucleotide will likely produce an RNA transcript that isat least partially identical in sequence to the transcript produced fromthe other desired polynucleotide.

The desired polynucleotide and its inverse complement may be separatedby a spacer DNA sequence, such as an intron, that is of any length. Itmay be desirable, for instance, to reduce the chance of transcribing theinverse complement copy of the desired polynucleotide from the opposingpromoter by inserting a long intron or other DNA sequence between the3′-terminus of the desired polynucleotide and the 5′-terminus of itsinverse complement. For example, in the case of pSIM717 (FIG. 3) thesize of the intron (“I”) may be lengthened so that the transcriptionalcomplex of P1 is unlikely to reach the sequence of the inversecomplement of gus-S before becoming interrupted or dislodged.Accordingly, there may be about 50, 100, 250, 500, 2000 or more than2000 nucleotides positioned between the sense and antisense copies ofthe desired polynucleotide.

A desired polynucleotide of the present invention, e.g., a “first” or“second” polynucleotide as described herein may share sequence identitywith all or at least part of a sequence of a structural gene orregulatory element. For instance, a first polynucleotide may sharesequence identity with a coding or non-coding sequence of a target geneor with a portion of a promoter of the target gene. In one embodiment,the polynucleotide in question shares about 100%, 99%, about 98%, about97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%,about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%,about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%,about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%,about 51%, about 50%, about 49%, about 48%, about 47%, about 46%, about45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%,about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%,about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about15%, or about 5% or at least about 1% sequence identity with a targetgene or target regulatory element, such as a target promoter.

For ease, the term “desired polynucleotide” as used herein is notlimiting but includes other terms used herein such as “firstpolynucleotide” and “second polynucleotide” or any polynucleotide thatis used in a construct of the present invention to reduce expression ofa target gene or sequence. Hence a “desired polynucleotide” may be afirst or second polynucleotide or both.

In a simpler form, a construct of the present invention does not containtwo copies of the polynucleotide but only one copy. Accordingly, thepolynucleotide is operably linked to promoters at both its 5′- and 3′termini. In this particular arrangement, RNA transcripts will beproduced that comprise sequences from each strand of the DNA duplex. Anexample of this arrangement is shown in FIG. 3 as pSIM772.

A terminator-free cassette may exist as an extrachromosomal DNA moleculein a cell or it may be integrated by any one of a variety of mechanismsinto the nucleus, chromosome, or other endogenous nucleic acid of thecell. If the terminator-free cassette is stably integrated into thegenome of the cell, then it may be possible to produce a cell line, cellculture, biological tissue, plant, or organism that comprises thecassette in subsequent cell or organism generations.

Expression of such a construct in a plant will reduce or preventexpression of gene(s) that display either shares sequence identity orinverse complementarity with at least part of a desired polynucleotide.The invention is not bound by any particular theory or mechanism, butthe transcripts may, directly or indirectly, affect the activity of aregulatory sequence, such as a promoter, that is normally associatedwith the expression of a target gene in a cell; or the transcript maynegatively affect the accumulation of a transcript that is endogenouslyproduced in the target cell. Accordingly, either or both of transcriptaccumulation and transcript translation may be altered by the activityof the transcript produced by the expression cassette of the presentinvention.

A plant of the present invention may be a monocotyledonous plant, forinstance, alfalfa, canola, wheat, turf grass, maize, rice, oat, barley,sorghum, orchid, iris, lily, onion, banana, sugarcane, and palm.Alternatively, the plant may be a dicotyledonous plant, for instance,potato, tobacco, tomato, avocado, pepper, sugarbeet, broccoli, cassava,sweet potato, cotton, poinsettia, legumes, alfalfa, soybean, pea, bean,cucumber, grape, brassica, carrot, strawberry, lettuce, oak, maple,walnut, rose, mint, squash, daisy, and cactus.

The effect of the RNA molecule, which is produced by a terminator-freeexpression cassette of the present invention, may be assessed bymeasuring, directly or indirectly, the target nucleic acid or proteinlevel in the cell or environment in which the expression cassette ispresent. Thus, the effect of an expression cassette of the presentinvention in downregulating, suppressing, reducing, or preventing oreliminating target gene expression may be identified by a reduction inthe amount of RNA transcript that is produced by the target gene, or areduction in the amount of target gene protein product, or both.

A desired polynucleotide of a terminator-free construct described hereinmay be identical to, or share sequence identity with different kinds ofDNA regions, such as (1) at least part of the sequence that encodes atarget transcript, (2) at least part of the intron of a gene thatencodes a target transcript, (3) at least part of the promoter of a genethat encodes a target transcript, (4) part of the terminator of a genethat encodes a target transcript, whereby the polynucleotide is not aterminator, (5) the 3′-untranslated region of a gene, and (6) the5′-untranslated region of a gene. One or more nucleotides of any one ofthese regions may be mutated, altered, or substituted to increasesequence identity with a target sequence or to otherwise increase orenhance silencing of the target sequence.

The location of the target sequence, therefore, may be in, but is notlimited to, (i) the genome of a cell; (ii) at least one RNA transcriptnormally produced in a cell; or (iii) in a plasmid, construct, vector,or other DNA or RNA vehicle. The cell that contains the genome or whichproduces the RNA transcript may be the cell of a bacteria, virus,fungus, yeast, fly, worm, plant, reptile, bird, fish, or mammal.

Hence, the target nucleic acid may be one that is normally transcribedinto RNA from a cell nucleus, which is then in turn translated into anencoding polypeptide. Alternatively, the target nucleic acid may notactually be expressed in a particular cell or cell type. For instance, atarget nucleic acid may be a genomic DNA sequence residing in a nucleus,chromosome, or other genetic material, such as a DNA sequence ofmitochondrial DNA. Such a target nucleic acid may be of, but not limitedto, a regulatory region, an untranslated region of a gene, or anon-coding sequence.

Alternatively, the target nucleic acid may be foreign to a host cell butis present or expressed by a non-host organism. For instance, a targetnucleic acid may be the DNA or RNA molecule endogenous to, or expressedby, an invading parasite, virus, or bacteria.

Furthermore, the target nucleic acid may be a DNA or RNA moleculepresent or expressed by a disease cell. For instance, the disease cellmay be a cancerous cell that expresses an RNA molecule that is notnormally expressed in the non-cancerous cell type.

In plants, the desired polynucleotide may share sequence identity with atarget nucleic acid that is responsible for a particular trait of aplant. For instance, a desired polynucleotide may produce a transcriptthat targets and reduces the expression of a polyphenol oxidase genetarget in a plant and, thereby, modifies one or more traits orphenotypes associated with black spot bruising. Similarly, a desiredpolynucleotide may produce a transcript that targets and reduces theexpression of a starch-associated R1 target nucleic acid orphosphorylase target nucleic acid in a plant, thereby modifying one ormore traits or phenotypes associated with cold-induced sweetening.

An expression cassette in a construct of the present invention may beflanked by one or more transfer-DNA (“T-DNA”) border sequences. Any ofthe expression cassettes described herein, for instance, may be insertedinto the T-DNA of an Agrobacterium-derived plasmid, such as a Ti plasmidfrom A. tumefaciens.

A border sequence may comprise a sequence that is similar to atraditional Agrobacterium T-DNA border sequence, but actually is asequence that is native to a plant, but which can facilitate transferand integration of one nucleic acid into another. For instance, suchplant-derived transfer-DNA (“P-DNA”) border sequences can be isolatedfrom potato (SEQ ID NO: 44), tomato (SEQ ID NOs: 45-46), pepper (SEQ IDNO: 47), alfalfa (SEQ ID NO: 48), barley (SEQ ID NO: 49), and rice (SEQID NO: 50) shown in the sequence table elsewhere in this application.

Accordingly, any one of the expression cassettes described herein may beinserted into a transfer-DNA that is delimited by such P-DNA bordersequences, which are capable of integrating the cassette into anothernucleic acid, such as a plant genome or plant chromosome.

Accordingly, an Agrobacterium plasmid, which contains an expressioncassette described herein that does not comprise a DNA region that isinvolved in 3-end formation and polyadenylation of an RNA transcript,may be stably integrated into the genome of a plant viaAgrobacterium-mediated transformation. The progeny of that transformedplant, therefore, will continue to express the transcripts associatedwith the expression cassette.

The promoters that are used to initiate transcription of the desiredpolynucleotide may be constitutive, tissue-preferred, or induciblepromoters or permutations thereof. “Strong” promoters, for instance,include the potato ubiquitin-7 and ubiquitin-3 promoters, and ubiquitinpromoters from maize, rice, and sugarcane. They also include the riceactin promoter, various rubisco small subunit promoters, rubiscoactivase promoters, and rice actin promoters. Good tissue-preferredpromoters that are mainly expressed in potato tubers include thepromoters of the granule-bound starch synthase and ADP glucosepyrophosphorylase genes. There are various inducible promoters, buttypically an inducible promoter can be a temperature-sensitive promoter,a chemically-induced promoter, or a temporal promoter. Specifically, aninducible promoter can be a Ha hsp17.7 G4 promoter, a wheat wcs120promoter, a Rab 16A gene promoter, an α-amylase gene promoter, a pin2gene promoter, or a carboxylase promoter.

Accordingly, to facilitate identification of a plant that has beensuccessfully transformed with a terminator-free expression cassette, itmay be desirable to include within the region delineated by thetransfer-DNA border sequences a selectable or screenable marker.Inclusion of a marker is a standard procedure in Agrobacterium-mediatedtransformation and is employed to make it possible to readily identifysuccessfully-transformed plant material. In the expression cassettesdepicted in FIGS. 1-4, for instance, the marker is hygromycinphosphtransferase (“hptII”), which confers hygromycin resistance to aplant that expresses that marker. In such cassettes, therefore, aterminator or DNA region that is involved in 3-end formation andpolyadenylation of an RNA transcript is operably linked to the hptIIgene sequence. Other selectable and screenable markers may be usedinstead of hptII.

EXAMPLES Example 1 Conventional Silencing Constructs

The efficacy of various silencing constructs was tested by targeting thebeta glucuronidase (gus) reporter gene operationally linked to thestrong constitutive promoter of figwort mosaic virus, designated here as“P1” (SEQ ID NO: 1). This test system is stringent because the gusprotein is highly stable. Thus, only relatively large reductions in gustranscripts result in phenotypically detectable reductions of gusprotein levels. Most silencing constructs contain at least one copy ofthe same 304-bp gus gene fragment (SEQ ID NO: 2), operably linked ineither the sense or antisense orientation to a strong constitutivepromoter and in some cases followed by the terminator of theAgrobacterium nopaline synthase gene. The silencing constructs wereinserted next to an expression cassette for the hygromycinphosphotransferase (hptII) selectable marker gene between the T-DNAborders of transformation vectors. Resulting vectors were used toretransform a tobacco plant that had been transformed before with aT-DNA containing an expression cassette for the gus gene (see also FIGS.1 and 2).

The following transformation vectors were produced to study the role ofa terminator element in conventional silencing constructs:

pSIM714: The negative control vector pSIM714, which does not contain asilencing construct.

pSIM718: Vector pSIM718, which contains a ‘sense’ gus gene fragmentoperably linked to the terminator of the nopaline synthase gene (SEQ IDNO: 3) that represents strategies described in, e.g., U.S. Pat. Nos.5,283,184 and 5,231,020. This vector contains the gus gene fragmentoperably linked in the sense orientation to the promoter and followed bythe terminator.

pSIM140: Vector pSIM140, which is identical to pSIM718 except that thesilencing construct does not contain a terminator.

pSIM755: Vector pSIM755, which contains a terminator-containing‘antisense’ construct that represents strategies described in, e.g.,U.S. Pat. Nos. 5,107,065 and 5,759,829. This vector contains the gusgene fragment operably linked in the sense orientation to the promoterand followed by the terminator.

pSIM758: Vector pSIM758, which is identical to pSIM755 except that thesilencing construct does not contain a terminator.

pSIM374: Vector pSIM374, which contains a terminator-containingconstruct that comprises both a sense and antisense gus gene fragmentand represents strategies described in, e.g., WO 99/53050A1. This vectorcontains two copies of the gus gene fragment, one in the senseorientation and the other one in the antisense orientation and separatedfrom each other by an intron, depicted in SEQ ID NO: 4, and insertedbetween promoter and terminator.

pSIM728 and 777: Vector pSIM728, which is identical to pSIM374 exceptthat the silencing construct does not contain a terminator. VectorpSIM777 is identical to pSIM728 except that the P2 promoter is at theother side of the expression cassette.

Binary vectors containing the various constructs were introduced intoAgrobacterium. Ten-fold dilutions of overnight-grown cultures of theresulting strains were grown for five to six hours, precipitated for 15minutes at 2,800 RPM, washed with MS liquid medium (PhytoTechnology, KS)supplemented with sucrose (3%, pH 5.7) and resuspended in the samemedium to 0.2 OD/600 nm. The suspension was then used to infect leafexplants of the transgenic in vitro grown Nicotiana tabacum (tobacco)plant expressing the gus gene. Infected explants were incubated for twodays on co-culture medium (1/10 MS salts, 3% sucrose, pH 5.7) containing6 g/L agar at 25° C. in a Percival growth chamber (16/8 hr photoperiod)and subsequently transferred to M401/agar (PhytoTechnology) mediumcontaining timentin (150 mg/L) and hygromycin (20 mg/L). Resultingshoots were transferred to hormone-free rooting medium, and three leavesof each resulting plant were stained for gus expression.

Table 1 shows that all plants retransformed with pSIM714 displayed thesame levels of gus expression as the original gus plant, confirming thatretransformation, proliferation of single cells, and regeneration doesnot negatively affect expression of the reporter gene.

Table 1 also shows that the constructs representing the three differentconventional silencing methods trigger gus gene silencing with varyingefficiencies. In agreement with what has been reported in theliterature, pSIM374 is most effective. About half of plants that wereretransformed with this constructs display at least some reduced levelof gus activity. The two other constructs support a reduction in gusactivity in only about 6% of retransformed plants.

Importantly, Table 1 also demonstrates that removal of the terminatordramatically lowers the efficacy of the silencing constructs. Forinstance, pSIM374 is more than six-fold more efficacious than itsterminator-free derivative, pSIM728. Hardly any activity is observedwith the terminator-free pSIM758.

It can be concluded that the terminator plays an essential role inoptimizing the activity of conventional silencing constructs.

Example 2 Effective Gene Silencing with Terminator-Free ConstructsComprising at Least Two Copies of a Target Gene Fragment that TriggerConvergent Transcription

The following transformation vectors were produced to study the effectof convergent transcription on gene silencing (see also FIG. 3):

pSIM715: Vector pSIM715 contains a construct that comprises a firstsegment consisting of the gus gene fragment operationally linked to thepromoter (P1) and a second segment in the opposite orientation thatconsists of the same gus gene fragment operationally linked to theconstitutive 35 S promoter of cauliflower mosaic virus, designated ‘P2’and depicted in SEQ ID NO: 5, whereby the first and second segment areseparated by two different introns.

pSIM717: Vector pSIM717 is identical to pSIM715 except that the twosegments of the construct are separated by a single intron.

pSIM789: Vector pSIM789 is identical to pSIM717 except that the P2promoter is replaced by a P1 promoter.

pSIM771: Vector pSIM771 is identical to pSIM717 except that the P1promoter is replaced by the potato ubiquitin-7 promoter, which isdepicted in SEQ ID NO: 6 and named here ‘P3’.

pSIM770: Vector pSIM770 is identical to pSIM717 except that the P2promoter that drives expression of the selectable marker gene isreplaced by P1, and the P1 promoter of the silencing construct isreplaced by P2.

pSIM772: Vector pSIM772 contains the gus gene fragment inserted betweentwo different oppositely oriented promoters, P2 and P3

pSIM756: Vector pSIM756 is identical to pSIM717 except that the gus genefragments are oriented in the inverse complementary orientation relativeto the promoter to which they are immediately linked.

pSIM779: Vector pSIM779 is an example of a tandem repeat of gus genefragments inserted between two convergent promoters.

pSIM787: Vector pSIM787 is similar as pSIM779 but contains four directrepeats of the target gene fragment inserted between convergentpromoters.

pSIM1111 is identical to pSIM779 except that the four direct repeats arepreceded by an antisense DNA fragment of the gus gene that is differentfrom SEQ ID NO: 2 and depicted as SEQ ID NO: 7.

Gus assays performed on re-transformed gus plants demonstrate that alltested terminator-free constructs that contain two segments, eachcontaining a different promoter driving the gus gene fragment, in theinverse complementary orientation, pSIM715, 717, and 756, 771 are moreefficaceous in silencing the gus gene than pSIM374, the construct thatrepresents the best conventional approach. Furthermore, pSIM789 alsoconfers effective gene silencing to many of the double transformants.

The experiment also shows that the use of a single gus gene fragment(pSIM779) is not as efficaceous. This result suggests that convergenttranscription of at least two copies of the desired polynucleotide isimportant for effective silencing.

The experiment also showed that a construct with two direct repeats(pSIM780) triggered gene silencing. However, this arrangement was not aseffective as the inverted repeat organization of pSIM756 (Table 1).Furthermore, four directs repeats (pSIM787) are more effective than twodirect repeats (Table 1).

To study the molecular basis of terminator-free silencing, RNA wasisolated from three plants that had been retransformed with pSIM717 andthree additional plants retransformed with pSIM715. In each case, oneplant represented an ineffective silencing event whereas the other twoplants displayed near-complete gus gene silencing.

Reverse-transcription polymerase chain reactions (RT-PCRs) wereperformed to study to production of transcripts from the two differentpromoters used in pSIM715 and pSIM717. The first primer used for theseexperiments (PG, shown in SEQ ID NO: 8) is specific for a sequence ofthe gus gene fragment and anneals to transcripts produced from eitherstrand. The second primer was designed to anneal to intron sequences ofone of the strands only (pIF, shown in SEQ ID NO: 9, anneals to asequence of the GBSS-intron derived spacer region of transcriptsproduced by the P1 promoter, and PIR, shown in SEQ ID NO: 10, anneals totranscripts produced by the P2 promoter). Interestingly, these studiesdemonstrated that the construct of the non-silenced plants 717-7 and717-13 only contained transcripts produced from one of the two strands,either T1 or T2 (FIG. 7). In contrast, the silenced plants 715-19,715-38, 717-55, and 717-36 produced transcripts from both strands (FIG.7). Thus, effective silencing is accomplished only if both promoters ofthe construct are functionally active simultaneously.

Hybridization of subsequent RNA gel blots with radioactively labeledprobes derived from the gus gene demonstrated that effective silencingin 715-38, 715-55, 717-12, 717-36, and 717-19 is correlated with astrong decrease in gus RNA accumulation (FIG. 8). Furthermore, thetranscripts produced by the silencing construct in fully silenced plantswere generally found to be varying in size from about 0.2-kb to about1.0-kb (FIG. 8). Although RT-PCR revealed the presence of additionallarge transcripts that comprise not only the polynucleotide but alsodownstream promoter sequences, the presence of such transcripts couldhardly be detected on RNA gel blots. For instance, hybridization with aprobe derived from the P1 promoter required a seven-day exposure timebefore an extremely faint smear could be observed.

Example 3 Silencing in Potato Tubers

Various vectors were developed to test the concept of terminator-freesilencing in tubers. These vectors contained an expression cassette forthe neomycin phosphotransferase (nptII) gene as selectable marker system(see also FIG. 6). The driver promoters used for gene silencing inpotato tubers were selected from the group consisting of: (1) the strongpotato ubiquitin-7 promoter, (2) the strong tuber and stolon-specificpromoter of the granule-bound starch synthase (GBSS) gene (SEQ ID NO:12), and (3) the strong tuber-specific promoter of the potato ADPglucose pyrophosphorylase (AGP) gene (SEQ ID NO: 13). See FIG. 4 formaps of the transfer DNAs.

pSIM764: Vector pSIM764 contains a ‘tuber-silencing’ construct thatcomprises a first segment consisting of the 154-bp trailer of the potatotuber-expressed PPO gene (SEQ ID NO: 14) operably linked to the GBSSpromoter and a second segment in the opposite orientation that consistsof the same trailer fragment operably linked to the GBSS promoterwhereby the first and second segment are separated by the intron of thepotato ubiquitin-7 gene depicted in SEQ ID NO: 15.

pSIM765: Vector pSIM765 is identical to pSIM764 except that the PPO genefragments are oriented in the opposite orientation.

pSIM217 represents the control plasmid and contains the two copies ofthe PPO gene inserted as inverted repeat between GBSS promoter andubiquitin terminator.

Ten-fold dilutions of overnight-grown cultures were grown for 5-6 hours,precipitated for 15 minutes at 2,800 RPM, washed with MS liquid medium(Phytotechnology) supplemented with sucrose (3%, pH 5.7), andresuspended in the same medium to 0.2 OD/600 nm. The resuspended cellswere mixed and used to infect 0.4-0.6 mm internodal segments of thepotato variety “Ranger Russet”. Infected stems were incubated for twodays on co-culture medium (1/10 MS salts, 3% sucrose, pH 5.7) containing6 g/L agar at 22° C. in a Percival growth chamber (16 hrs light) andsubsequently transferred to callus induction medium (CIM, MS mediumsupplemented with 3% sucrose 3, 2.5 mg/L of zeatin riboside, 0.1 mg/L ofnaphthalene acetic acid, and 6 g/L of agar) containing timentin (150mg/L) and kanamycin (100 mg/L). After one month of culture on CIM,explants were transferred to shoot induction medium (SIM, MS mediumsupplemented with 3% sucrose, 2.5 mg/L of zeatin riboside, 0.3 mg/L ofgiberellic acid GA3, and 6 g/L of agar) containing timentin andkanamycin (150 and 100 mg/L respectively) until shoots arose. Shootsarising at the end of regeneration period were transferrred to MS mediumwith 3% sucrose, 6 g/L of agar and timentin (150 mg/L). Transgenicplants were transferred to soil and placed in a growth chamber (11 hourslight, 25° C.). After three weeks, at least 3 minitubers/line wereassayed for PPO activity. For this purpose, 1 g of potato tubers ispulverized in liquid nitrogen, added to 5 ml of 50 mM MOPS(3-(N-morpholino) propane-sulfonic acid) buffer (pH 6.5) containing 50mM catechol, and incubated at room temperature with rotation for about 1hour. The solid fraction was precipitated, and the supernatanttransferred to another tube to determine PPO activity. For this purpose,1 g of potato tubers was pulverized in liquid nitrogen. This powder wasthen added to 5 ml of 50 mM MOPS (3-(N-morpholino) propane-sulfonicacid) buffer (pH 6.5) containing 50 mM catechol, and incubated at roomtemperature with rotation for about 1 hour. The solid fraction was thenprecipitated, and the supernatant transferred to another tube todetermine PPO activity by measuring the change of OD-410 over time. Theexperiment demonstrated that pSIM764 and 765 trigger effective silencingin potato tubers (Table 2). A comparison with data presented in WO2003/069980 demonstrates that the method of the present invention can bemore effective than that of conventional terminator-based gene silencingas exemplified by pSIM217, the PPO control.

Example 4 Multi-Gene Silencing in Tobacco

Two constructs were created to study the effect of the position of genefragments within the silencing construct. For this purpose, the twocopies of the gus gene fragment of pSIM771 were replaced by two copiesof the gus gene fragment linked to a fragment of the tobacco polyphenoloxidase (PPO) gene (SEQ ID NO: 16) (also, see FIG. 6):

pSIM774: Vector pSIM774 contains a silencing construct with the gus genefragments immediately linked to the promoters and the adjacent PPO genefragments linked to the central intron.

pSIM775: Vector pSIM775 contains a silencing construct with the PPO genefragments immediately linked to the promoters and the adjacent gus genefragments linked to the central intron.

Retransformation of gus plants with these vectors is expected to triggersilencing as efficiently as pSIM771.

Example 5 Multi-Gene Silencing in Potato

Multiple gene silencing is implemented by simultaneously targeting threeundesirable potato tuber genes.

Plasmid pSIM1121 (Russet Boise II) comprises an all-native transfer DNAdepicted in SEQ ID NO: 17 comprising a silencing construct comprisingtwo copies of a DNA segment, separated by the intron of the potatoubiquitin-7 gene and positioned as inverted repeat between twoconvergent GBSS promoters, whereby the DNA segment comprises (i) afragment of the trailer of the tuber-expressed polyphenol oxidase geneof the wild potato relative Solanum verrucosum Schltdl. TRHRG 193,accession number 498062 (see: USDA, ARS, National Genetic ResourcesProgram. Germplasm Resources Information Network—(GRIN). [OnlineDatabase] National Germplasm Resources Laboratory, Beltsville, Md.Available:http://www.ars-grin.gov2/cgi-bininpgs/html/acchtml.pl?1392998, 12 Sep.2005) (SEQ ID NO: 18) (ii) a fragment of the leader of the phosphorylaseL gene (SEQ ID NO: 19), and (iii) a fragment of the leader of the R1gene (SEQ ID NO: 20).

Employment of this plasmid makes it possible to produce transformedpotato plants that only contain native DNA and display the following newtraits: (1) bruise tolerance due to silencing of the tuber-expressed PPOgene, (2) reduced cold-induced glucose accumulation due to silencing ofthe phosphorylase and R1 genes.

Example 6 Highly Effective Promoter Targeting

The following transformation vectors were produced to demonstrate thatsequences of the target promoter can be used to silence expression ofthe target gene (see also FIG. 4):

pSIM773: Vector pSIM773 contains a construct that comprises a firstsegment comprising the P3 promoter linked to P1, and a second segment,which is oriented in the opposite orientation, and which comprises theP2 linked to P1. The first and second segment are separated by anintron. Thus, this construct contains four functionally activepromoters. The two promoters in the middle are identical, represent thetarget promoter, and are in convergent orientation. The two outsidepromoters are different to each other and in convergent orientation. Allfour promoters contain a TATA box and proceed up to a base pair upstreamfrom the transcription start.

pSIM1101: Vector pSIM1101 is identical to pSIM773 except that the P3promoter was replaced by the nos terminator.

pSIM788: Vector pSIM788 is similar to pSIM773 except that the twocentral P1 promoters of the target gus gene only contain sequencesupstream from the TATA box, (SEQ ID NO: 21), thus representingnon-functional promoters.

pSIM1120: Vector pSIM1120 is similar to pSIM773 except that the twocentral promoters of the target gene lack a TATA box and are not inconvergent but divergent orientation.

pSIM1112: Vector pSIM1112 contains a single non-functional P1 promoterinserted between convergent P2 and P3 promoter.

pSIM1113: Vector pSIM1113 contains two convergent P1 promoters separatedby an intron.

pSIM754: Control vector pSIM754 contains the P1 promoter drivingexpression of the P2 promoter, and vice versa.

Retransformation of gus plants with pSIM773 yielded 35 hygromycinresistant plants. PCR analysis confirmed the presence of the transferDNA of pSIM773. Surprisingly, subsequent gus staining revealed anextremely effective complete silencing of the gus gene (Table 1). Twentyplants (57%) did not display any detectable gus expression. Thus,promoter targeting using the pSIM773 strategy is highly desirable.

Similar results were obtained with the target promoters in divergentorienteation inserted between two convergent driver promoters, with 77%of plants that had been retransformed with pSIM1120 displaying full gusgene silencing (Table 1).

Table 1 shows that gene silencing was also accomplished by using asingle target promoter inserted between two convergent driver promoters(pSIM1112). However, this method may be less effective than methods thatemploy two copies of the target promoter oriented as inverted repeat.

Furthermore, efficacy of pSIM1113 demonstrates that driver promoters arenot always necessary. It is possible to effectively silencing a gene bysimply employing two convergent target promoters (Table 1).

Many (44%) of the plants that were retransformed with pSIM1101 alsodisplayed full gene silencing (Table 1). This finding demonstrates thatpromoter-based silencing does not require convergent transcription.

Conventional silencing methods have often been found to not providestable gene silencing in subsequent generations. In contrast,four-promoter constructs represented by pSIM773 gave full silencing thatis completely maintained upon transmission of the silencing cassette tothe next generation. The enhanced stability was demonstrated by allowingdouble transformed tobacco plants to mature, and subsequentlydetermining gus expression levels in T1 progenies. This study showedthat 100% of the progeny plants that were derived from a pSIM773 plantand contained both gus gene and silencing cassette displayed full gusgene silencing (Table 3). In contrast, none of the T1 plants carryingthe gus gene and pSIM374 silencing cassette displayed full gus genesilencing (Table 3). An intermediair phenotype was observed by analyzingthe progeny of a plant carrying the gus gene and the silencing cassetteof pSIM717 (Table 3).

Example 7 Sequence Requirements for Promoter Targeting

The above experiments demonstrated that promoter sequences can be usedto effectively trigger gene silencing. However, they should not beunderstood to imply that any promoter fragment of the target gene couldbe employed for this purpose.

To study the sequence requirements for promoter-based silencing, twovectors were created that comprise two copies of only part of the P1promoter inserted as inverted repeat between the driver promoters.

pSIM1118: Vector pSIM1118 contains two copies of an upstream 300-bpfragment of the promoter shown in SEQ ID NO: 11.

pSIM1119: Vector pSIM1119 contains two copies of a central 300-bp regionof the P1 promoter shown in SEQ ID NO: 51.

Retransformation of gus plants with the two different constructs yielded34 and 20 plants, respectively, that were analyzed histochemically.Interestingly, none of the analyzed plants displayed any reduced gusexpression, indicating that the employed promoter fragments did noteffectively trigger gene silencing (Table 1).

FIG. 9 shows a sequence analysis of the various promoter fragments. Thefragment that facilitates effective gene silencing is present inpSIM773, 788, 1101, and 1120 but not in pSIM1118 and 1119.

Example 8 Reduced Cold-Sweetening in Tubers of Potato Plants Containinga Silencing Construct Comprising Two Copies of a Fragment of thePromoter of the R1 Gene

The sequence of the promoter of the potato starch-associated R1 gene,including leader and start codon, is shown in SEQ ID NO: 22. Two copiesof a short (342-bp) R1 promoter fragment (SEQ ID NO: 23) were insertedas inverted repeat between either two convergently oriented promoters ofthe GBSS promoter (in plasmid pSIM1038) or a GBSS and AGP promoter inconvergent orientation (in plasmid pSIM1043). The resulting binaryvectors were used to produce transformed potato plants. These plantswill be allowed to develop tubers, and the tubers will be stored forabout a month or longer at 4° C. Glucose analysis of the cold-storedtubers will demonstrate that the transformed plants accumulate lessglucose than untransformed control plants. The reduced accumulation ofglucose will lower color formation during French fry processing and,thus, make it possible to reduce blanch time and preserve more of theoriginal potato flavor. Furthermore, promoter-mediated R1 gene silencingwill limit starch phosphorylation and, therefore, reduce theenvironmental issues related to the release of waste water containingpotato starch. Other benefits of the transformed tubers include: (1)resulting French fries will contain lower amounts of the toxic compoundacrylamide, which is formed through a reaction between glucose andasparagine, and (2) resulting fries will display a crisper phenotype, asevaluated by professional sensory panels, due to the slightly alteredstructure of the starch.

Similar results can be obtained by employing a shorter (151-bp) part ofthe R1 promoter, shown in SEQ ID NO. 24. Binary vector pSIM1056comprises two copies of this fragment inserted as inverted repeatbetween two convergently oriented GBSS promoters; pSIM1062 comprises thefragments inserted between convergently oriented GBSS and AGP promoters.This vector was used to produce 25 transformed plants, which can beshown to display reduced cold-induced glucose accumulation and allbenefits associated with that trait.

Example 9 Enhanced Blackspot Bruise Tolerance in Tubers of Potato PlantsContaining a Silencing Construct Comprising Two Copies of a Fragment ofthe Promoter of the Polyphenol Oxidase Gene

The sequence of the promoter of the potato tuber-expressed polyphenoloxidase gene is shown in SEQ ID NO: 25. Two copies of a 200-bp PPOpromoter fragment (SEQ ID NO: 26) were inserted as inverted repeatbetween convergent GBSS and AGP promoters. A binary vector comprisingthis silencing construct, designated pSIM1046, was used to producetwenty-five transformed potato plants. The plants can be allowed todevelop tubers, and the tubers can be assayed for polyphenol oxidaseactivity. Such an analysis will show that the expression level of thetargeted PPO gene is reduced if compared to levels in untransformedcontrols.

In a similar way, plasmid pSIM1045, which contains two copies of a460-bp PPO promoter fragment (SEQ ID NO: 27) inserted between convergentGBSS and AGP promoters, can be used to lower PPO gene expression.

Similar strategies can be used in other crop species to limit bruise.For instance, the promoter of the leaf-expressed PPO gene of lettuce canbe used to reduce bruise in lettuce leaves, the promoter of thefruit-expressed PPO gene of apple can be used to reduce bruise in applefruit, and the promoter of the seed-expressed PPO gene of wheat can beused to reduce bruise in wheat grains. In all these and other cases, thepromoter can be isolated straightforwardly by designing primers thatanneal to the known PPO gene sequences, and performing well-known DNAisolation methods such as inverse PCR.

Example 10 Improved Oil Content in Seeds of Canola Plants Containing aSilencing Construct Comprising Two Copies of a Fragment of the Promoterof the Fad2 Gene

The sequence of the promoter of the Brassica Fad2 gene, includingleader, intron, and start codon, is shown in SEQ ID NO: 28. Two copiesof a fragment of this promoter lacking any transcribed sequences such asthe 441-bp fragment shown in SEQ ID 29 can be placed as inverted repeatbetween two convergently oriented promoters that are expressed inBrassica seeds. Examples of ‘driver’ promoters are: the promoter of anapin (1.7S seed storage protein gene) gene shown in SEQ ID NO: 30 orthe promoter of a stearoyl-ACP desaturase gene (SEQ ID NO: 31).

The silencing cassette can be placed within the transfer DNA sequence ofa binary vector, and this binary vector can be used to transformBrassica. Some of the resulting plants will produce seed that containsincreased amounts of oleic acid.

Other promoters that can be used in silencing constructs to improve oilcomposition in oilseed crops such as canola, soybean, cotton, andsunflower include promoters of other genes of the fatty acidbiosynthesis pathway. For instance, a promoter of a target fatty aciddesaturase 12, or microsomal omega-6 fatty acid desaturase, (FAD12) gene(e.g., Genbank Accession Nr. AF243045 for canola and AB188250 forsoybean) such as the soybean FAD12 promoter shown in SEQ ID NO: 32 canbe used to increase oleic acid levels in crops such as canola andsoybean.

Furthermore, promoters of the cotton stearoyl-acyl-carrier protein delta9-desaturase and oleoyl-phosphatidylcholine omega 6-desaturase genes canbe used to increase stearic acid and oleic acid levels, respectively, incotton. This promoter can be identified by performing methods such asinverse PCR using the known sequence of the target genes (Liu et al.,Plant Physiol 129:1732-43, 2002). Two copies of the newly isolatedpromoter can then be used in strategies similar to that shown forpSIM773 whereby the ‘driver’ seed-specific promoters can eitherrepresent foreign DNA or native DNA.

Example 11 Reduced Lignin Content in the Vascular System of AlfalfaPlants Containing a Silencing Construct Comprising Two Copies of aFragment of the Promoter of the Comt Gene

The promoter of the Medicago sativa (alfalfa) caffeicacid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) gene,including leader, is shown in SEQ ID NO: 33.

Two copies of a 448-bp promoter fragment that lacks transcribedsequences (SEQ ID NO: 34) were inserted as inverted repeat between twoconvergently oriented driver promoters. The first driver promoter is thepromoter of the petE gene shown in SEQ ID NO: 35; the second promoter isthe promoter of the Pal gene shown in SEQ ID NO: 36. A binary vectorcomprising this silencing construct, designated pSIM1117, was used toproduce transformed alfalfa plants. Stem tissues of the plants wereassayed and shown to contain reduced levels of lignin.

Reduced lignin content can be determined according to the followingprotocol: (i) cut stem sections and place them on watch glass, (ii)immerse the cut stems in 1% potassium permanganate for 5 min at roomtemperature, (iii) discard the potassium permanganate solution using adisposable pipette and wash the samples twice with water to removeexcess potassium permanganate, (iv) add 6% HC1 (V/V) and let the colorof the sections turn from black or dark brown to light brown, (v) ifnecessary, add additional HC1 to facilitate the removal of dark color,(vi) discard the HC1 and wash the samples twice with water, (vii) addfew drops of 15% sodium bicarbonate solution (some times it may not gointo solution completely), a dark red or red-purple color develops forhardwoods (higher in S units) and brown color for softwood (higher in Gunits).

Nineteen transformed alfalfa lines were tested for reduced lignincontent, and six plants were found to accumulate reduced amounts of theS-unit of lignin.

Instead of the promoter of the COMT gene, it is also possible to use thepromoter of the caffeoyl CoA 3-O-methyltransferase (CCOMT) gene. Thesequence of this promoter, together with downstream leader, is shown inSEQ ID NO: 37. A fragment of SEQ ID NO: 29 that lacks transcribedsequences as depicted in SEQ ID NR: 38 can be used to lower lignincontent.

Similarly, lignin can be reduced in trees by using promoters of genesinvolved in lignin biosynthesis. It is also possible to use SEQ ID NO:59 and reduce lignin content in maize by employing the above-describedpromoter-based silencing approach.

Example 12 Increased Shelf Life of Fruits of Tomato Plants Containing aSilencing Construct Comprising Two Copies of a Fragment of the Promoterof the Polygalacturonase Gene

A promoter of a target polygalacturonase gene such as the tomatopromoter shown in SEQ ID NO: 39 can be used to reduce breakdown ofpectin, thus slowing cell wall degradation, delaying softening,enhancing viscosity characteristics, and increasing shelf life in tomatoby inserting two copies of the promoter fragment as inverted repeatbetween convergent fruit-specific driver promoters.

Example 13 Reduced Allergenicity of Foods from Plants Containing aSilencing Construct Comprising Two Copies of a Fragment of the Promoterof Genes Encoding Allergens

The promoter of the major apple allergen Mal d 1 gene can be isolated byemploying inverse PCR methods using the known gene sequence (Gilissen etal., J Allergy Clin Immunol 115:364-9, 2005), and this promoter can thenbe used to develop apple varieties that contain lower allergenicitylevels.

Similarly, the promoter of the major peanut allergen Ara h 2 (Dodo etal., Curr Allergy Asthma Rep 5, 67-73, 2005) can be isolated usinginverse PCR methods, and used to develop peanut varieties that containlower allergenicity levels.

Furthermore, the promoter of the major soybean allergen Gly m Bd 30 K(Herman et al., Plant Physiol 132, 36-43, 2003) can be isolated usinginverse PCR methods, and used to develop peanut varieties that containlower allergenicity levels.

Example 14 Multi-Gene Silencing Approach Based on a Combination of Geneand Promoter Fragments

Plasmid pSIM870 (Russet Boise III) comprises an all-native transfer DNAdepicted in SEQ ID NO. 40 comprising (1) a first silencing cassettecomprising two copies of a DNA segment positioned as inverted repeatbetween two convergent GBSS promoters whereby the DNA segment comprises(i) a fragment of the trailer of the tuber-expressed polyphenol oxidasegene of Solanum verrucosum, (ii) a fragment of the leader of thephosphorylase L gene, and (iii) a fragment of the trailer of thephosphorylase L gene (SEQ ID NO: 41), and (2) a second silencingcassette comprising two copies of the R1 promoter positioned as invertedrepeat between the driver promoters of the AGP and GBSS genes,respectively.

Plasmid pSIM899 (Russet Boise IV) comprises an all-native transfer DNAdepicted in SEQ ID NO: 42 comprising a first silencing cassettecomprising two copies of a DNA segment positioned as inverted repeatbetween two convergent GBSS promoters whereby the DNA segment comprises(i) a fragment of the trailer of the tuber-expressed polyphenol oxidasegene of Solanum verrucosum, and (ii) a fragment of the leader of thephosphorylase L gene, and a second silencing cassette comprising fourcopies of the leader of the R1 gene operably linked to the AGP promoterand followed by an inverted repeat comprising a sense and antisensefragment of the R1 gene.

Potato transformation with any of these three plasmids will produceplants that, compared to untransformed plants, display the followingcharacteristics: (1) reduced expression of the tuber-expressedpolyphenol oxidase gene and, consequently, (i) increased tuberpolyphenol content as can be determined by xx, and (ii) enhancedtolerance to tuber black spot bruise as can be determined by xx, and (2)strongly reduced expression of the phosphorylase and R1 genes and,consequently, (i) reduced starch phosphorylation and, consequently,lowered phosphate content of waste waters containing potato starch, and(ii) a reduced conversion of starch into glucose during cold-storage asdetermined by using the glucose oxidase/peroxidase reagent (Megazyme,Ireland), resulting in (a) less caramelization, and consequently,reduced color formation during frying, which makes it possible to storeat higher temperatures and/or blanch for shorter time periods (b) lessformation of acrylamide, and (c) increased crispness of fries.

Example 15 Intron Targeting

The polynucleotide used to generate a TFCT construct can contain theintron of a gene that produces the target transcript. The concept ofintron-targeted silencing can be demonstrated by using the intron of thegus gene that is expressed in transgenic tobacco.

The following transformation vector was produced to demonstrate thatsequences of the target intron can be used to silence expression of thetarget gene (see also FIG. 5):

Vector pSIM782, which contains a construct that comprises a firstsegment consisting of the intron of the gus gene operationally linked tothe promoter (P1) and a second segment in the opposite orientation thatconsists of the same gus gene intron operationally linked to a secondconstitutive promoter (P2) whereby the first and second segment areseparated by an intron.

An example of an intron that can be used to silence a gene is the intronof the Solanum vernei starch-associated R1 gene SEQ ID NO: 44. R1 genesilencing will reduce the extent of cold-induced sweetening in tubersduring storage.

Example 16 Terminator Targeting

The polynucleotide used to generate a TFCT construct can comprisesequences downstream from the transcribed sequences of a target gene.This concept can be demonstrated by using the sequences downstream fromthe gus gene that is expressed in transgenic tobacco.

Example 17 Reduced Lignin Content in the Vascular System of AlfalfaPlants Containing a Silencing Construct Comprising Two Copies of aFragment of the Comt Gene

A binary vector designated pSIM856 was assembled comprising anexpression cassette comprising two Comt gene fragments depicted in SEQID NOs: 52 and 53, positioned as inverted repeat between two convergentalfalfa promoters shown in SEQ ID NOs: 54 and 55 in such a way that thepromoters are operably linked to first the antisense fragment and thenthe sense fragment. The expression cassette is inserted between alfalfaderived sequences that function as replacement for Agrobacterium bordersand are shown in SEQ ID NOs: 56 and 57. The entire transfer DNA,depicted in SEQ ID NO: 58 is inserted into a plasmid that carries anexpression cassette for the Agrobacterium ipt gene in its backbone.

Transformations were carried out as described in Weeks and Rommens, USpatent application US20050034188A1, which is incorporated herein byreference. Two transformed plants were tested for lignin content, andboth were found to not visibly accumulate the S-unit.

Tables

TABLE 1 Efficacy of conventional and terminator-free silencingconstructs. Tobacco Construct for 2^(nd) plants gus expressiontransformation assayed 50-100% 10-50% 1-10% 0% none 3 3 (100%) 0 0 0PSIM714 8 8 (100%) 0 0 0 PSIM374 36 13 (36%) 11 (31%) 9 (25%) 3 (8%)PSIM718 35 33 (95%) 1 (3%) 1 (3%) 0 PSIM728 23 15 (65%) 5 (22%) 3 (13%)0 PSIM715 37 10 (27%) 11 (30%) 15 (41%) 1 (3%) PSIM717 35 11 (31%) 3(9%) 19 (54%) 2 (6%) pSIM754 38 38 (100%) 0 0 0 PSIM755 36 35 (97%) 0 01 (3%) pSIM756 37 18 (49%) 12 (32%) 5 (14%) 2 (5%) PSIM758 29 29 (100%)0 0 0 PSIM770 38 35 (92%) 3 (8%) 0 0 PSIM771 35 20 (57%) 3 (9%) 9 (26%)3 (9%) PSIM772 35 34 (97%) 0 1 (3%) 0 PSIM773 35 15 (43%) 0 0 20 (57%)PSIM774 35 31 (89%) 2 (6%) 2 (6%) 1 (3%) PSIM775 36 22 (61%) 6 (17%) 7(19%) 1 (3%) PSIM777 36 33 (92%) 1 (3%) 1 (3%) 1 (3%) PSIM778 36 32(89%) 2 (6%) 2 (6%) 0 PSIM779 36 33 (92%) 1 (3%) 2 (6%) 0 PSIM782 35 34(97%) 0 1 (3%) 0 PSIM787 32 20 (63%) 7 (22%) 3 (9%) 2 (6%) PSIM788 35 14(40%) 0 0 21 (60%) PSIM789 35 19 (54%) 4 (11%) 6 (17%) 6 (17%) PSIM110134 14 (41%) 0 5 (15%) 15 (44%) PSIM1111 36 21 (58%) 9 (25%) 6 (17%) 0pSIM1112 36 33 (92%) 1 (3%) 0 2 (6%) pSIM1113 34 24 (71%) 2 (6%) 3 (9%)5 (15%) PSIM1118 34 34 (100%) 0 0 0 PSIM1119 20 20 (100%) 0 0 0 pSIM112035 8 (23%) 0 0 27 (77%)

TABLE 2 PPO activity in potato mini tubers. rep-1(OD) rep-2 (OD) rep-3(OD) % of WT S.E. Control wt-1 0.127 0.121 0.137 87 2.6 wt-2 0.129 0.1410.125 89 2.7 wt-3 0.138 0.146 0.123 92 3.7 wt-4 0.134 0.157 0.159 1014.4 wt-5 0.152 0.173 0.169 111 3.6 wt-6 0.153 0.152 0.151 103 0.3 wt-70.173 0.158 0.167 112 2.4 wt-8 0.149 0.165 0.152 105 2.7 401-1 0.1380.155 0.174 105 5.7 401-2 0.182 0.193 0.163 121 4.8 401-3 0.139 0.1450.152 98 2.1 pSIM764 1 0.051 0.055 0.060 37 1.4 2 0.071 0.072 0.068 480.7 3 0.063 0.070 0.075 47 1.9 4 0.035 0.032 0.030 22 0.8 5 0.045 0.0310.030 24 2.7 6 0.053 0.056 0.056 37 0.6 7 0.079 0.108 0.117 68 6.3 80.035 0.042 0.041 27 1.2 9 0.039 0.042 0.043 28 0.7 10 0.081 0.073 0.07752 1.3 11 0.059 0.061 0.052 39 1.5 12 0.055 0.046 0.053 35 1.5 13 0.0360.039 0.032 24 1.1 14 0.052 0.068 0.062 41 2.6 15 0.037 0.033 0.034 230.7 16 0.066 0.057 0.066 43 1.7 17 0.063 0.061 0.057 41 1.0 18 0.0630.041 0.047 34 3.6 18 0.045 0.049 0.041 30 1.3 20 0.061 0.051 0.048 362.2 21 0.043 0.039 0.039 27 0.7 22 0.111 0.102 0.112 73 1.8 23 0.0580.049 0.057 37 1.6 24 0.043 0.041 0.042 28 0.3 25 0.041 0.040 0.045 280.8 26 0.044 0.042 0.042 29 0.4 PSIM765 1 0.044 0.035 0.039 27 1.4 20.041 0.048 0.055 32 2.2 3 0.064 0.060 0.058 41 1.0 5 0.122 0.118 0.10277 3.4 10 0.042 0.066 0.059 38 3.9 14 0.087 0.103 0.111 68 3.9 15 0.0450.049 0.059 34 2.3 16 0.033 0.042 0.035 25 1.5 19 0.033 0.048 0.045 282.5 20 0.043 0.040 0.052 30 2.0 21 0.044 0.035 0.033 25 1.9 24 0.0460.049 0.047 32 0.5 28 0.046 0.048 0.033 29 2.6 29 0.071 0.082 0.078 521.8 30 0.051 0.059 0.056 37 1.3 32 0.105 0.134 0.129 83 4.9 34 0.0450.047 0.038 29 1.5 35 0.143 0.168 0.171 109 4.9 36 0.115 0.128 0.097 775.0 37 0.057 0.049 0.040 33 2.7 38 0.062 0.067 0.063 43 0.8 39 0.0460.055 0.045 33 1.8 40 0.040 0.036 0.036 25 0.7 41 0.083 0.069 0.072 502.3 ‘wt’ = untransformed wild type plants; ‘401’ = transformed plantscarrying a transfer DNA only comprising an expression cassette for thenptII seelectable marker gene, ‘OD’ = OD260 measurement, ‘S.E.’ =standard error.

TABLE 3 PCR positive for both gus gene and Parental line silencingconstruct Partially silenced Fully silenced 374-18 25/50 (50%) 24/25(96%) 0 717-54 35/50 (70%) 28/35 (80%) 3/35 (9%)  773-4 23/50 (46%) 023/23 (100%)

1-13. (canceled)
 14. A construct, comprising an expression cassettewhich comprises in the 5′ to 3′ orientation (i) a promoter, (ii) a firstpolynucleotide that comprises a sequence that shares sequence identitywith at least a part of a promoter sequence associated with a targetgene, (iii) a second polynucleotide comprising a sequence that is aperfect or imperfect inverse complement of the first polynucleotide, and(iv) a terminator, wherein the first promoter is operably linked to the5′-end of the first polynucleotide and the second polynucleotide isoperably linked to the terminator. 15-32. (canceled)
 33. The constructof claim 14, wherein the first polynucleotide and the promoter sequenceassociated with the target gene are fully identical in sequence over atleast 23 nucleotides.
 34. The construct of claim 14, wherein the secondpolynucleotide and the first polynucleotide do not comprise any sequencelocated downstream of the transcription start of the target gene. 35.The construct of claim 14, wherein (a) at least part of the firstdesired polynucleotide is in the antisense orientation; or (b) at leastpart of the first desired polynucleotide is in the sense orientation.36. The construct of claim 14, further comprising a spacerpolynucleotide positioned between the first and second polynucleotides,wherein the spacer polynucleotide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or more than 500 nucleotides long.
 37. The construct of claim 14,wherein the promoters and the terminator are functional in a plant andwherein the expression cassette is located between transfer-DNA bordersequences of a plasmid that is suitable for bacterium-mediated planttransformation, wherein the bacterium is a strain of Agrobacterium,Rhizobium, or Phyllobacterium.
 38. The construct of claim 14, whereinthe promoter is a constitutive promoter, a near-constitutive promoter, atissue-specific promoter, or an inducible promoter.
 39. The construct ofclaim 14, wherein the promoter is a functional plant promoter specificto a plant tissue selected from the group consisting of in tubers,seeds, leaves, roots, vascular system, flowers, pollen, and ovules. 40.The construct of claim 14, wherein the target gene is a COMT geneinvolved in lignin biosynthesis, a CCOMT gene involved in ligninbiosynthesis, any other gene involved in lignin biosynthesis, an R1 geneinvolved in starch phosphorylation, a phosphorylase gene involved instarch phosphorylation, a PPO gene involved in oxidation of polyphenols,a polygalacturonase gene involved in pectin degradation, a gene involvedin the production of allergens, or a gene involved in fatty acidbiosynthesis.
 41. The construct of claim 14, wherein the promotersequence associated with the target gene is selected from the groupconsisting of (1) a starch-associated R1 gene promoter, (2) a polyphenoloxidase gene promoter, (3) a fatty acid desaturase 12 gene promoter, (4)a microsomal omega-6 fatty acid desaturase gene promoter, (5) a cottonstearoyl-acyl-carrier protein delta 9-desaturase gene promoter, (6) anoleoyl-phosphatidylcholine omega 6-desaturase gene promoter, (7) aMedicago truncatula caffeic acid/5-hydroxyferulic acid3/5-O-methyltransferase (COMT) gene promoter, (8) a Medicago sativa(alfalfa) caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase(COMT) gene promoter, (9) a Medicago truncatula caffeoyl CoA3-O-methyltransferase (CCOMT) gene promoter, (10) a Medicago sativa(alfalfa) caffeoyl CoA 3-O-methyltransferase (CCOMT) gene promoter, (11)a Zea mays (maize) COMT gene, (12) a major apple allergen Mal d 1 genepromoter, (13) a major peanut allergen Ara h 2 gene promoter, (14) amajor soybean allergen Gly m Bd 30 K gene promoter, (15) apolygalacturonase gene promoter, (16) any other endogenous promoter. 42.A method for downregulating the expression of a target gene in a plantcell, comprising expressing the construct of claim 14 in a plant cell,wherein the expression level of the target gene is downregulatedcompared to the expression level of the target gene in an untransformedplant cell.
 43. A method for enhancing tolerance to black spot bruisingin a tuber, comprising expressing the construct of claim 14 in a cell ofa tuber, wherein (a) the first polynucleotide comprises the sequencethat shares sequence identity with at least a part of a tuber-expressedpolyphenol oxidase gene promoter, (b) the promoter is GBSS or AGP, and(c) expression of the construct in the cell reduces transcription and/ortranslation of a polyphenol oxidase gene in the tuber cell genome,thereby enhancing the tolerance of the tuber to black spot bruising. 44.A method for reducing cold-induced sweetening in a tuber, comprisingexpressing the construct of claim 14 in a cell of a tuber, wherein (a)the first polynucleotide comprises the sequence that shares sequenceidentity with at least a part of an R1 gene promoter, (b) the promotersis GBSS or AGP, and (c) expression of the construct in the cell reducestranscription and/or translation of an R1 gene in the tuber cell genome,thereby reducing cold-induced sweetening in the tuber.
 45. A method forincreasing oleic acid levels in an oil-bearing plant, comprisingexpressing the construct of claim 14 in a cell of a seed of anoil-bearing plant, wherein (a) the first polynucleotide comprises asequence that shares sequence identity with at least a part of a Fad2gene promoter, (b) the promoter is a napin gene promoter, a Fad2 genepromoter, or a stearoyl-ACP desaturase gene promoter, and (c) expressionof the construct in the cell reduces transcription and/or translation ofa Fad2 gene in the cell of the seed of the oil-bearing plant, therebyincreasing the oil content of the seed.
 46. A method for reducing lignincontent in a plant, comprising expressing the construct of claim 14 in acell of the plant, wherein (a) the first polynucleotide comprises asequence that shares sequence identity with at least part of thesequence of the promoter associated with a gene selected from the groupconsisting of caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase(COMT) gene and caffeoyl CoA 3-O-methyltransferase (CCOMT) gene, (b) thepromoter is a petE or Pal gene promoter, and (c) expression of theconstruct in the cell reduces transcription and/or translation of a COMTor CCOMT gene in the cell of the plant, thereby reducing lignin contentin a plant.
 47. A method for reducing the degradation of pectin in afruit of a plant, comprising expressing the construct of claim 14 in afruit cell of the plant, wherein (a) the first polynucleotide comprisesa sequence that shares sequence identity with at least a part of apolygalacturonase gene promoter, (b) the promoter is a fruit-specificpromoter, and (c) expression of the construct in the fruit cell reducestranscription and/or translation of a polygalacturonase gene in the cellof the plant, thereby reducing the degradation of pectin in the fruit.48. A method for reducing the allergenicity of a food produced by anapple plant, comprising expressing the construct of claim 14 in a cellof the apple plant, wherein (a) the first polynucleotide comprises asequence from the Mal d I gene promoter, and (b) expression of theconstruct in the apple plant reduces transcription and/or translation ofMal d I in the apple.
 49. A method for reducing the allergenicity of afood produced by a peanut plant, comprising expressing the construct ofclaim 14 in a cell of the peanut plant, wherein (a) the firstpolynucleotide comprises a sequence from the Ara h 2 gene promoter, and(b) expression of the construct in the peanut plant reducestranscription and/or translation of Ara h 2 in the peanut.
 50. A methodfor reducing the allergenicity of a food produced by a soybean plant,comprising expressing the construct of claim 14 in a cell of the soybeanplant, wherein (a) the first polynucleotide comprises a sequence fromthe Gly m Bd gene promoter, and (b) expression of the construct in thesoybean plant reduces transcription and/or translation of Gly m Bd inthe soybean.
 51. A plant cell obtained according to the method of claim42.
 52. A plant comprising the plant cell of claim
 51. 53. A foodproduct made from the plant of claim 52.